BBA - Molecular Basis of Disease (v.1812, #3)

The role of CDX2 in intestinal homeostasis and inflammation by Mehmet Coskun; Jesper Thorvald Troelsen; Ole Haagen Nielsen (283-289).
Many transcription factors are known to control transcription at several promoters, while others are only active at a few places. However, due to their importance in controlling cellular functions, aberrant transcription factor function and inappropriate gene regulation have been shown to play a causal role in a large number of diseases and developmental disorders. Inflammatory bowel disease (IBD) is characterized by a chronically inflamed mucosa caused by dysregulation of the intestinal immune homeostasis. The aetiology of IBD is thought to be a combination of genetic and environmental factors, including luminal bacteria. The Caudal-related homeobox transcription factor 2 (CDX2) is critical in early intestinal differentiation and has been implicated as a master regulator of the intestinal homeostasis and permeability in adults. When expressed, CDX2 modulates a diverse set of processes including cell proliferation, differentiation, cell adhesion, migration, and tumorigenesis. In addition to these critical cellular processes, there is increasing evidence for linking CDX2 to intestinal inflammation. The aim of the present paper was to review the current knowledge of CDX2 in regulation of the intestinal homeostasis and further to reveal its potential role in inflammation.► CDX2 regulates intestinal homeostasis. ► Altered CDX2 expression triggers inflammation. ► CDX2 is a downstream target of inflammatory cytokines.
Keywords: CDX2; IBD; Inflammation; Intestine; MAPK; NF-κB;

Essential role of neutrophil mobilization in concanavalin A-induced hepatitis is based on classic IL-6 signaling but not on IL-6 trans-signaling by Sven Malchow; Wolfgang Thaiss; Nathalie Jänner; Georg H. Waetzig; Jessica Gewiese-Rabsch; Christoph Garbers; Kosuke Yamamoto; Stefan Rose-John; Jürgen Scheller (290-301).
Neutrophil depleted mice are protected from concanavalin A-mediated hepatitis, showing that neutrophils are critical for cellular liver damage. Interleukin-6 has pro- and anti-inflammatory properties and mediates neutrophil recruitment in diseases such as rheumatoid arthritis. In classic signaling, interleukin-6 binds to the membrane-bound interleukin-6-receptor and initiates signaling via gp130. In interleukin-6 trans-signaling, the agonistic soluble interleukin-6-receptor can form a soluble interleukin-6/interleukin-6-receptor complex and stimulate cells which only express gp130 but no interleukin-6-receptor. Interleukin-6 trans-signaling was shown to be important for liver regeneration and development of liver adenomas. Here, we show that blocking classic interleukin-6 signaling but not interleukin-6 trans-signaling reduced concanavalin A-induced liver damage in mice, with reduced liver STAT3 phosphorylation and liver neutrophil accumulation. However, the level of neutrophil-attracting chemokine KC is only reduced by inhibition of interleukin-6 trans-signaling. Analysis of circulating neutrophils after concanavalin A challenge revealed that classic interleukin-6 signaling is required for the mobilization of blood neutrophils. Reduced neutrophil infiltration was accompanied by increased levels of hepatoprotective monocyte chemoattractant protein-1 and reduced level of hepatodestructive interleukin-4. Abrogated classic interleukin-6 signaling in concanavalin A-mediated hepatitis exhibited liver-protective effects indicating that interleukin-6 classic but not interleukin-6 trans-signaling is responsible for liver damage. Classic interleukin-6 signaling is required to mount an efficient neutrophilia during concanavalin A-induced immune response, which might have clinical implications in the regard that blocking global interleukin-6 signaling pathways is a treatment option in different chronic inflammatory diseases.►Blocking classic IL-6 signaling reduced ConA-induced liver damage. ►Blocking IL-6 trans-signaling has no effect on ConA-induced liver damage. ►IL-6 blockade led to reduced liver infiltrating neutrophils. ►Classic IL-6 signaling is required for the mobilization of blood neutrophils. ►IL-6 blockade led to increased MCP-1 and reduced IL-4.
Keywords: Interleukin-6; ConA; Hepatitis; Liver damage; Neutrophilia;

Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxia-induced cell death by Nicolle Bristow; Teralee R. Burton; Elizabeth S. Henson; Coleen Ong-Justiniano; Michelle Brown; Spencer B. Gibson (302-311).
BNIP3 (Bcl-2/adenovirus E1B Nineteen Kilodalton Interacting Protein) is a pro-cell death member of the Bcl-2 family of proteins. Its expression is induced by the transcription factor Hypoxia Inducible Factor-1 (HIF-1) under conditions of low oxygen (hypoxia) and is found over expressed in hypoxic regions of many tumors. When over expressed, BNIP3 induces cell death through induction of mitochondrial dysfunction that is dependent on the presence of BNIP3's TM domain. Herein, we have determined that the SkOv3 ovarian cancer cell line expresses a truncated BNIP3 protein, which results in the elimination of the transmembrane domain. Truncation that eliminates all four domains of BNIP3 protein also inhibits hypoxia-induced cell death in SkOv3, HEK293, U251 and MCF-7 cells. Three different mutations in a BNIP3 expression vector that lead to a truncated BNIP3 protein, lacking TM domain only, or lacking CD, BH3, and TM domains resulted in inhibition of hypoxia-induced cell death when transfected into HEK293 cells. We found that truncated BNIP3 failed to associate with the mitochondria and the truncated BNIP3 lacking all four domains can bind to wild type BNIP3. Taken together, truncation of BNIP3 could be a novel mechanism for cancer cells to avoid hypoxia-induced cell death mediated by BNIP3 over expression.► Truncated BNIP3 lacks all four domains found in the wild type protein. ► Truncated BNIP3 protein inhibits hypoxia-induced cell death. ► Truncated BNIP3 failed to associate with the mitochondria. ► Truncated BNIP3 lacking all four domains can bind to wild type BNIP3. ► Truncated BNIP3 can prevent wild type BNIP3 localization to the mitochondria.
Keywords: Bcl-2 family; Hypoxia; Normoxia; BNIP3; Cell death and cancer;

Primary carnitine deficiency is caused by impaired activity of the Na+-dependent OCTN2 carnitine/organic cation transporter. Carnitine is essential for entry of long-chain fatty acids into mitochondria and its deficiency impairs fatty acid oxidation. Most missense mutations identified in patients with primary carnitine deficiency affect putative transmembrane or intracellular domains of the transporter. Exceptions are the substitutions P46S and R83L located in an extracellular loop close to putative glycosylation sites (N57, N64, and N91) of OCTN2. P46S and R83L impaired glycosylation and maturation of OCTN2 transporters to the plasma membrane. We tested whether glycosylation was essential for the maturation of OCTN2 transporters to the plasma membrane. Substitution of each of the three asparagine (N) glycosylation sites with glutamine (Q) decreased carnitine transport. Substitution of two sites at a time caused a further decline in carnitine transport that was fully abolished when all three glycosylation sites were substituted by glutamine (N57Q/N64Q/N91Q). Kinetic analysis of carnitine and sodium-stimulated carnitine transport indicated that all substitutions decreased the Vmax for carnitine transport, but N64Q/N91Q also significantly increased the Km toward carnitine, indicating that these two substitutions affected regions of the transporter important for substrate recognition. Western blot analysis confirmed increased mobility of OCTN2 transporters with progressive substitutions of asparagines 57, 64 and/or 91 with glutamine. Confocal microscopy indicated that glutamine substitutions caused progressive retention of OCTN2 transporters in the cytoplasm, up to full retention (such as that observed with R83L) when all three glycosylation sites were substituted. Tunicamycin prevented OCTN2 glycosylation, but it did not impair maturation to the plasma membrane. These results indicate that OCTN2 is physiologically glycosylated and that the P46S and R83L substitutions impair this process. Glycosylation does not affect maturation of OCTN2 transporters to the plasma membrane, but the 3 asparagines that are normally glycosylated are located in a region important for substrate recognition and turnover rate.► Natural mutations P46S and R83 L in the OCTN2 transporter affect glycosylation. ► Identification of natural glycosylation sites of the OCTN2 carnitine transporter. ► Characterization of the effects of OCTN2 glycosylation on membrane maturation. ► Characterization of the effects of OCTN2 glycosylation on transport activity.
Keywords: Primary carnitine deficiency; Fatty acid oxidation; OCTN2; SLC22A5; Organic cation transporter; Carnitine transport; Glycosylation;

POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts by Joanna D. Stewart; Susanne Schoeler; Kamil S. Sitarz; Rita Horvath; Kerstin Hallmann; Angela Pyle; Patrick Yu-Wai-Man; Robert W. Taylor; David C. Samuels; Wolfram S. Kunz; Patrick F. Chinnery (321-325).
Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.► The restoration of mitochondrial DNA (mtDNA) levels following ethidium bromide depletion was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. ► These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells. ► This approach provides a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.
Keywords: Mitochondria; Mitochondrial DNA; Depletion; Ethidium bromide;

Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia, an inborn error of bone and teeth metabolism associated with reduced levels of serum alkaline phosphatase activity. A missense mutation (c.346G>A) of TNSALP gene, which converts Ala to Thr at position 116 (according to standardized nomenclature), was reported in dominantly transmitted hypophosphatasia patients (A.S. Lia-Baldini et al. Hum Genet. 109 (2001) 99–108). To investigate molecular phenotype of TNSALP (A116T), we expressed it in the COS-1 cells or Tet-On CHO K1 cells. TNSALP (A116T) displayed not only negligible alkaline phosphatase activity, but also a weak dominant negative effect when co-expressed with the wild-type enzyme. In contrast to TNSALP (W, wild-type), which was present mostly as a non-covalently assembled homodimeric form, TNSALP (A116T) was found to exist as a monomer and heterogeneously associated aggregates covalently linked via disulfide bonds. Interestingly, both the monomer and aggregate forms of TNSALP (A116T) gained access to the cell surface and were anchored to the cell membrane via glycosylphosphatidylinositol (GPI). Co-expression of secretory forms of TNSALP (W) and TNSALP (A116T), which are engineered to replace the C-terminal GPI anchor with a tag sequence (his-tag or flag-tag), resulted in the release of heteromeric complexes consisting of TNSALP (W)-his and TNSALP (A116T)-flag. Taken together, these findings strongly suggest that TNSALP (A116T) fails to fold properly and forms disulfide-bonded aggregates, though it is indeed capable of interacting with the wild-type and reaching the cell surface, therefore explaining its dominant transmission.► TNSALP (W) monomer assembles into non-covalently associated homodimer. ► TNSALP (A116T) fails to form non-covalent homodimer. ► TNSALP (A116T) forms disulfide-bonded aggregates. ► The aggregate of TNSALP (A116T) traps TNSALP (A116T), suggestive of dominant negative inhibition.
Keywords: Dominance; Genetic disease; Hypophosphatasia; Inborn error of metabolism; Tissue-nonspecific alkaline phosphatase;

Preface: Zebrafish Models of Neurology by W. Ted Allison (333-334).

Zebrafish models for the functional genomics of neurogenetic disorders by Edor Kabashi; Edna Brustein; Nathalie Champagne; Pierre Drapeau (335-345).
In this review, we consider recent work using zebrafish to validate and study the functional consequences of mutations of human genes implicated in a broad range of degenerative and developmental disorders of the brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish homologs of human genes and to directly express human genes bearing mutations related to neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.► Expression of human genes in zebrafish. ► Genetics of degenerative brain disorders in zebrafish. ► Genetics of degenerative spinal cord disorders in zebrafish. ► Genetics of neurodevelopmental disorders in zebrafish. ► Future prospects for zebrafish molecular genetics and disease modeling.
Keywords: Zebrafish; Human diseases; Neurogenetic diseases; Neurodegenerative disorders; Neurodevelopmental diseases; Animal models; Genomics; Human genetics; Trangenic animals;

Zebrafish as a tool in Alzheimer's disease research by Morgan Newman; Giuseppe Verdile; Ralph N. Martins; Michael Lardelli (346-352).
Alzheimer's disease is the most prevalent form of neurodegenerative disease. Despite many years of intensive research our understanding of the molecular events leading to this pathology is far from complete. No effective treatments have been defined and questions surround the validity and utility of existing animal models. The zebrafish (and, in particular, its embryos) is a malleable and accessible model possessing a vertebrate neural structure and genome. Zebrafish genes orthologous to those mutated in human familial Alzheimer's disease have been defined. Work in zebrafish has permitted discovery of unique characteristics of these genes that would have been difficult to observe with other models. In this brief review we give an overview of Alzheimer's disease and transgenic animal models before examining the current contribution of zebrafish to this research area. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.► The molecular pathology of Alzheimer's disease is still poorly understood. ► Zebrafish possess orthologues of human genes involved in familial and sporadic Alzheimer's disease. ► Zebrafish embryos are a powerful tool for molecular biological analyses of Alzheimer's disease molecular pathology.
Keywords: Zebrafish; Alzheimer's disease; Presenilin;

Zebrafish models of Tauopathy by Qing Bai; Edward A. Burton (353-363).
Tauopathies are a group of incurable neurodegenerative diseases, in which loss of neurons is accompanied by intracellular deposition of fibrillar material composed of hyperphosphorylated forms of the microtubule-associated protein Tau. A zebrafish model of Tauopathy could complement existing murine models by providing a platform for genetic and chemical screens, in order to identify novel therapeutic targets and compounds with disease-modifying potential. In addition, Tauopathy zebrafish would be useful for hypothesis-driven experiments, especially those exploiting the potential to deploy in vivo imaging modalities. Several considerations, including conservation of specialized neuronal and other cellular populations, and biochemical pathways implicated in disease pathogenesis, suggest that the zebrafish brain is an appropriate setting in which to model these complex disorders. Novel transgenic zebrafish lines expressing wild-type and mutant forms of human Tau in CNS neurons have recently been reported. These studies show evidence that human Tau undergoes disease-relevant changes in zebrafish neurons, including somato-dendritic relocalization, hyperphosphorylation and aggregation. In addition, preliminary evidence suggests that Tau transgene expression can precipitate neuronal dysfunction and death. These initial studies are encouraging that the zebrafish holds considerable promise as a model in which to study Tauopathies. Further studies are necessary to clarify the phenotypes of transgenic lines and to develop assays and models suitable for unbiased high-throughput screening approaches. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.► Tauopathies are common, incurable neurodegenerative diseases. ► A zebrafish Tauopathy model might allow chemical and genetic screens to isolate novel treatment approaches. ► Structural and biochemical conservation suggest that the zebrafish CNS is an appropriate setting to model disease. ► Transgenic zebrafish expressing human Tau show features of human Tauopathy.
Keywords: Tau; Tauopathy; Progressive supranuclear palsy; Zebrafish; Alzheimer's disease; Transgenic; Neurodegeneration;

Zebrafish possess a robust, innate CNS regenerative ability. Combined with their genetic tractability and vertebrate CNS architecture, this ability makes zebrafish an attractive model to gain requisite knowledge for clinical CNS regeneration. In treatment of neurological disorders, one can envisage replacing lost neurons through stem cell therapy or through activation of latent stem cells in the CNS. Here we review the evidence that radial glia are a major source of CNS stem cells in zebrafish and thus activation of radial glia is an attractive therapeutic target. We discuss the regenerative potential and the molecular mechanisms thereof, in the zebrafish spinal cord, retina, optic nerve and higher brain centres. We evaluate various cell ablation paradigms developed to induce regeneration, with particular emphasis on the need for (high throughput) indicators that neuronal regeneration has restored sensory or motor function. We also examine the potential confound that regeneration imposes as the community develops zebrafish models of neurodegeneration. We conclude that zebrafish combine several characters that make them a potent resource for testing hypotheses and discovering therapeutic targets in functional CNS regeneration. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.► We review the robust innate regenerative potential of the zebrafish CNS. ► Radial glia have been identified as a major source of CNS stem cells. ► We examine the molecular pathways activated during regenerative processes. ► Innovative cell lesioning paradigms that induce regeneration are described. ► Emerging genetic and physiology tools recommend zebrafish for CNS regeneration studies.
Keywords: Zebrafish; Regeneration; Retina; Optic nerve; Spinal cord; Brain; Central nervous system; Stem cell; Glia;

Altered neurological function will generally be behaviourally apparent. Many of the behavioural models pioneered in mammalian models are portable to zebrafish. Tests are available to capture alterations in basic motor function, changes associated with exteroceptive and interoceptive sensory cues, and alterations in learning and memory performance. Excepting some endpoints involving learning, behavioural tests can be carried out at 4 days post fertilization. Given larvae can be reared quickly and in large numbers, and that software solutions are readily available from multiple vendors to automatically test behavioural responses in 96 larvae simultaneously, zebrafish are a potent and rapid model for screening neurological impairments. Coupling current and emerging behavioural endpoints with molecular techniques will permit and accelerate the determination of the mechanisms behind neurotoxicity and degeneration, as well as provide numerous means to test remedial drugs and other therapies. The emphasis of this review is to highlight unexplored/underutilized behavioural assays for future studies. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.►Neurotoxicity and neurodegeneration typically have associated behavioural phenotypes. ►Zebrafish behavioural assays have tight parallel with mammalian assays. ►Behavioural assays can test motor and sensory neuron function, and cognitive performance. ►Numerous behavioural assays can be conducted on zebrafish 4 days post fertilization
Keywords: Zebrafish; Behaviour; Activity; Sensorimotor; Learning endpoints;

Aberrant forebrain signaling during early development underlies the generation of holoprosencephaly and coloboma by Patricia A. Gongal; Curtis R. French; Andrew J. Waskiewicz (390-401).
In this review, we highlight recent literature concerning the signaling mechanisms underlying the development of two neural birth defects, holoprosencephaly and coloboma. Holoprosencephaly, the most common forebrain defect, occurs when the cerebral hemispheres fail to separate and is typically associated with mispatterning of embryonic midline tissue. Coloboma results when the choroid fissure in the eye fails to close. It is clear that Sonic hedgehog (Shh) signaling regulates both forebrain and eye development, with defects in Shh, or components of the Shh signaling cascade leading to the generation of both birth defects. In addition, other intercellular signaling pathways are known factors in the incidence of holoprosencephaly and coloboma. This review will outline recent advances in our understanding of forebrain and eye embryonic pattern formation, with a focus on zebrafish studies of Shh and retinoic acid pathways. Given the clear overlap in the mechanisms that generate both diseases, we propose that holoprosencephaly and coloboma can represent mild and severe aspects of single phenotypic spectrum resulting from aberrant forebrain development. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.►Holoprosencephaly is caused by alterations in Shh or RA signaling. ►Coloboma is caused by alterations in Shh or BMP signaling. ►Coloboma and Holoprosencephaly are part of a mutational spectrum.
Keywords: Holoprosencephaly; Coloboma; Sonic hedgehog; Gdf6; Retinoic acid; Retina;

Fish models in prion biology: Underwater issues by Edward Málaga-Trillo; Evgenia Salta; Antonio Figueras; Cynthia Panagiotidis; Theodoros Sklaviadis (402-414).
Transmissible spongiform encephalopathies (TSEs), otherwise known as prion disorders, are fatal diseases causing neurodegeneration in a wide range of mammalian hosts, including humans. The causative agents – prions – are thought to be composed of a rogue isoform of the endogenous prion protein (PrP). Beyond these and other basic concepts, fundamental questions in prion biology remain unanswered, such as the physiological function of PrP, the molecular mechanisms underlying prion pathogenesis, and the origin of prions. To date, the occurrence of TSEs in lower vertebrates like fish and birds has received only limited attention, despite the fact that these animals possess bona fide PrPs. Recent findings, however, have brought fish before the footlights of prion research. Fish models are beginning to provide useful insights into the roles of PrP in health and disease, as well as the potential risk of prion transmission between fish and mammals. Although still in its infancy, the use of fish models in TSE research could significantly improve our basic understanding of prion diseases, and also help anticipate risks to public health. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.►Prion diseases may possibly be transmitted to fish species through the food chain. ►Fish and mammalian PrPs share important cellular roles in cell–cell communication. ►The use of zebrafish in prion research may help elucidating the role of PrP in health and disease and also addressing TSE-related issues concerning public health.
Keywords: Prion protein; Transmissible spongiform encephalopathy; Neurodegeneration; Fish; Zebrafish;

The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type specific manner. In this review, reggie microdomains, lipid rafts, related scaffolding proteins and caveolin—which, however, are responsible for their own microdomains and functions—are introduced. Moreover, the function of the reggies in axon growth is demonstrated: neurons fail to extend axons after reggie knockdown. Furthermore, our current concept of the molecular mechanism underlying reggie function is presented: the association of glycosyl-phophatidyl inositol (GPJ)-anchored surface proteins with reggie microdomains elicits signals which activate src tyrosine and mitogen-activated protein kinases, as well as small guanosine 5′-triphosphate-hydrolyzing enzymes. This leads to the mobilization of intracellular vesicles and to the recruitment of bulk membrane and specific cargo proteins, such as cadherin, to specific sites of the plasma membrane such as the growth cone of elongating axons. Thus, reggies regulate the targeted delivery of cargo—a process which is required for process extension and growth. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.► Lipid raft-associated proteins. ► Caveolin. ► SPFH-family. ► Regulation of membrane protein trafficking.
Keywords: Axon regeneration; Microdomain; Reggie/flotillin; Recruitment; Targeted delivery;