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Behavioral phenotypes of amyloid-based genetically modified mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative affliction of the elderly, presenting with progressive memory loss and dementia and terminating with death. There have been significant advances in understanding the biology and subsequent diagnosis of AD; however, the furious pace of research has not yet translated into a disease-modifying treatment. While scientific inquiry in AD is largely centered on identifying biological players and pathological mechanisms, the day-to-day realities of AD patients and their caregivers revolve around their steady and heartbreaking cognitive decline. In the past decade, AD research has been fundamentally transformed by the development of genetically modified animal models of amyloid-driven neurodegeneration. These important in vivo models not only replicate some of the hallmark pathology of the disease, such as plaque-like amyloid accumulations and astrocytic inflammation, but also some of the cognitive impairments relevant to AD. In this article, we will provide a detailed review of the behavioral and cognitive deficits present in several transgenic mouse models of AD and discuss their functional changes in response to experimental treatments.

Alzheimer Disease↗

Mutation in TRMU related to transfer RNA modification modulates the phenotypic expression of the deafness-associated mitochondrial 12S ribosomal RNA mutations.

The human mitochondrial 12S ribosomal RNA (rRNA) A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, it has been proposed that nuclear-modifier genes modulate the phenotypic manifestation of the A1555G mutation. Here, we identified the nuclear-modifier gene TRMU, which encodes a highly conserved mitochondrial protein related to transfer RNA (tRNA) modification. Genotyping analysis of TRMU in 613 subjects from 1 Arab-Israeli kindred, 210 European (Italian pedigrees and Spanish pedigrees) families, and 31 Chinese pedigrees carrying the A1555G or the C1494T mutation revealed a missense mutation (G28T) altering an invariant amino acid residue (A10S) in the evolutionarily conserved N-terminal region of the TRMU protein. Interestingly, all 18 Arab-Israeli/Italian-Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited prelingual profound deafness. Functional analysis showed that this mutation did not affect importation of TRMU precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNA. As a consequence, these defects contribute to the impairment of mitochondrial-protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA mutations.

Amino Acid Sequence↗

Mitochondrial 12S rRNA A827G mutation is involved in the genetic susceptibility to aminoglycoside ototoxicity.

We have analyzed the clinical and molecular characterization of a Chinese family with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluations revealed that only those family members who had a history of exposure to aminoglycoside antibiotics subsequently developed hearing loss, suggesting mitochondrial genome involvement. Sequence analysis of the mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, a mutation that was identified previously in a few sporadic patients and in another Chinese family with non-syndromic deafness. The pathogenicity of the A827G mutation is strongly supported by the occurrence of the same mutation in two independent families and several genetically unrelated subjects. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly conserved in mammals. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. However, incomplete penetrance of hearing impairment indicates that the A827G mutation itself is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Indeed, aminoglycosides may contribute to the phenotypic manifestation of the A827G mutation in this family. In contrast with the congenital or early-onset hearing impairment in another Chinese family carrying the A827G mutation, three patients in this pedigree developed hearing loss only after use of aminoglycosides. This discrepancy likely reflects the difference of genetic backgrounds, either mitochondrial haplotypes or nuclear modifier genes, between two families.

Aminoglycosides↗

Chemically modified dextrans modulate expression of collagen phenotype by cultured smooth muscle cells in relation to the degree of carboxymethyl, benzylamide, and sulfation substitutions.

We developed regenerating agents (RGTAs) corresponding to polysaccharides derived from dextran and containing defined amounts of carboxymethyl (CM), carboxymethyl sulfate (CMS), carboxymethyl benzylamide (CMB), or carboxymethyl benzylamide sulfate (CMBS) groups with varying degrees of substitution. These compounds mimicked some effects of heparin on smooth muscle cell (SMC) proliferation and promoted in vivo tissue remodeling. We demonstrated that only RGTAs containing both CM and sulfate groups decreased SMC proliferation, in correlation with increased sulfation level. This effect was amplified by the presence of benzylamide. Independent of this activity on cell proliferation (i.e., with postconfluent cells), RGTAs modulated collagen biosynthesis by SMCs. On the one hand, CMBS more than CMS RGTAs induced a decrease of collagen III synthesis at the level of mRNA steady state and protein production. On the other hand, CMS to a greater extent than CMBS RGTAs increased both collagen V mRNA and protein production. In addition, only benzylamide-containing RGTAs increased accumulation of collagen I and III in the cell layer. In conclusion, RGTA bioactivities required the presence of CM functions, increased with the sulfation level, and varied with benzylamide substitution. RGTAs that modulate cell proliferation and collagen biosynthesis by differential mechanisms may represent potential antifibrotic agents.

Animals↗

Maternally inherited non-syndromic hearing loss associated with mitochondrial 12S rRNA A827G mutation in a Chinese family.

We explored the clinical and molecular characterization of a Chinese family with non-syndromic hearing impairment. Clinical evaluations revealed a possible maternal inheritance pattern, and showed an extremely similar phenotype of hearing loss including the age of onset, severity, and audiometric configuration. Sequence analysis of the mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, which was absent in other family members and 40 Chinese controls. This mutation has previously been reported sporadically in a few individuals with aminoglycoside-induced and non-syndromic hearing loss. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly evolutionarily conserved in mammals. The occurrence of the A827G mutation in these genetically unrelated subjects strongly suggests that this mutation is involved in the pathogenesis of hearing impairment. However, incomplete penetrance of hearing loss indicates that the A827G mutation alone is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression, even though aminoglycosides and GJB2 gene may not contribute to the penetrance of the A827G mutation in this Chinese family. In contrast with the variable phenotype of hearing loss associated with other mitochondrial mutations, all of the patients in our family exhibited strikingly similar clinical features. This discrepancy likely reflects the difference of genetic backgrounds between this pedigree and others.

Asian People↗

Cowpea mosaic virus-based chimaeras. Effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses.

Expression of foreign peptides on the surface of cowpea mosaic virus particles leads to the creation of chimaeras with a variety of phenotypes and yields. Two factors were shown to be particularly significant in determining the properties of a given chimaera: the length of the inserted sequence and its isoelectric point. The deleterious effect of high isoelectric point on the ability of chimeras to produce a systemic infection occurs irrespective of the site of insertion of the peptide. Ultrastructural analysis of tissue infected with chimaeras with different phenotypes showed that all produced particles with a tendency to aggregate, irrespective of the size or isoelectric point of the insert. Host range and transmission studies revealed that the expression of a foreign peptide did not (1) alter the virus host range, (2) increase the rate of transmission by beetles or through seed, or (3) change the insect vector specificity. These findings have implications for both the utility and the biosafety of Cowpea mosaic virus-based chimaeras.

Amino Acid Sequence↗

Self-selection by genetically modified committed lymphocyte precursors reverses the phenotype of JAK3-deficient mice without myeloablation.

Janus kinase 3 (JAK3) is an essential component of cytokine receptor signal transduction pathways required for normal lymphocyte development and function. JAK3 deficiency in both mice and humans results in severe combined immunodeficiency (SCID) and increased susceptibility to opportunistic infections. We have previously shown that JAK3 gene transfer into irradiated recipients could restore immune function. However, since this toxic conditioning would be undesirable for infants in a clinical application, we have tested whether immune function could be restored in nonmyeloablated JAK3-deficient (-/-) mice. Murine JAK3 retroviral vectors were transduced into hematopoietic stem cells from the livers of newborn JAK3(-/-) mice. These cells were then injected intraperitoneally into nonirradiated JAK3(-/-) neonates. Transduced cells were detectable in these mice at time points 4 to 6 months after injection and resulted in significant correction of T and B lymphocyte numbers and circulating immunoglobulin (Ig) levels. After immune challenge with a dose of influenza A virus that was lethal to nonmanipulated JAK3(-/-) mice, mice injected with transduced cells showed development of circulating virus-specific IgG and enhanced survival. This work shows that the large selective advantage for JAK3-corrected lymphoid cells may be sufficient to overcome the need for myeloablative conditioning in JAK3 gene therapy protocols.

Animals↗

A genetic screen for dominant modifiers of a small-wing phenotype in Drosophila melanogaster identifies proteins involved in splicing and translation.

Studies in the fly, Drosophila melanogaster, have revealed that several signaling pathways are important for the regulation of growth. Among these, the insulin receptor/phosphoinositide 3-kinase (PI3K) pathway is remarkable in that it affects growth and final size without disturbing pattern formation. We have used a small-wing phenotype, generated by misexpression of kinase-dead PI3K, to screen for novel mutations that specifically disrupt organ growth in vivo. We identified several complementation groups that dominantly enhance this small-wing phenotype. Meiotic recombination in conjunction with visible markers and single-nucleotide polymorphisms (SNPs) was used to map five enhancers to single genes. Two of these, nucampholin and prp8, encode pre-mRNA splicing factors. The three other enhancers encode factors required for mRNA translation: pixie encodes the Drosophila ortholog of yeast RLI1, and RpL5 and RpL38 encode proteins of the large ribosomal subunit. Interestingly, mutations in several other ribosomal protein-encoding genes also enhance the small-wing phenotype used in the original screen. Our work has therefore identified mutations in five previously uncharacterized Drosophila genes and provides in vivo evidence that normal organ growth requires optimal regulation of both pre-mRNA splicing and mRNA translation.

Amino Acid Sequence↗

A new modified live equine influenza virus vaccine: phenotypic stability, restricted spread and efficacy against heterologous virus challenge.

Flu Avert IN vaccine is a new, live attenuated virus vaccine for equine influenza. We tested this vaccine in vivo to ascertain 1) its safety and stability when subjected to serial horse to horse passage, 2) whether it spread spontaneously from horse to horse and 3) its ability to protect against heterologous equine influenza challenge viruses of epidemiological relevance. For the stability study, the vaccine was administered to 5 ponies. Nasal swabs were collected and pooled fluids administered directly to 4 successive groups of naïve ponies by intranasal inoculation. Viruses isolated from the last group retained the vaccine's full attenuation phenotype, with no reversion to the wild-type virus phenotype or production of clinical influenza disease. The vaccine virus spread spontaneously to only 1 of 13 nonvaccinated horses/ponies when these were comingled with 39 vaccinates in the same field. For the heterologous protection study, a challenge model system was utilised in which vaccinated or naïve control horses and ponies were exposed to the challenge virus by inhalation of virus-containing aerosols. Challenge viruses included influenza A/equine-2/Kentucky/98, a recent representative of the 'American' lineage of equine-2 influenza viruses; and A/equine-2/Saskatoon/90, representative of the 'Eurasian' lineage. Clinical signs among challenged animals were recorded daily using a standardised scoring protocol. With both challenge viruses, control animals reliably contracted clinical signs of influenza, whereas vaccinated animals were reliably protected from clinical disease. These results demonstrate that Flu Avert IN vaccine is safe and phenotypically stable, has low spontaneous transmissibility and is effective in protecting horses against challenge viruses representative of those in circulation worldwide.

Administration, Intranasal↗

Genetic interactions of Drosophila melanogaster arrest reveal roles for translational repressor Bruno in accumulation of Gurken and activity of Delta.

arrest mutants have pleiotropic phenotypes, ranging from an early arrest of oogenesis to irregular embryonic segmentation defects. One function of arrest is in translational repression of oskar mRNA; this biochemical activity is presumed to be involved in other functions of arrest. To identify genes that could provide insight into how arrest contributes to translational repression or that may be targets for arrest-dependent translational control, we screened deficiency mutants for dominant modification of the arrest phenotype. Only four of the many deficiencies tested, which cover approximately 30% of the genome, modified the starting phenotype. One enhancer, identified fortuitously, is the Star gene. Star interaction with arrest results in excess Gurken protein, supporting the model that gurken is a target of repression. Two modifiers were mapped to individual genes. One is Lk6, which encodes a protein kinase predicted to regulate the rate-limiting initiation factor eIF4E. The second is Delta. The interaction between arrest and Delta mimics the phenotype of homozygous Delta mutants, suggesting that arrest could positively control Delta activity. Indeed, arrest mutants have significantly reduced levels of Delta protein at the interface of germline and follicle cells.

Animals↗

Genetic modification of glaucoma associated phenotypes between AKXD-28/Ty and DBA/2J mice.

BACKGROUND: Glaucoma is a common disease but its molecular etiology is poorly understood. It involves retinal ganglion cell death and optic nerve damage that is often associated with elevated intraocular pressure. Identifying genes that modify glaucoma associated phenotypes is likely to provide insights to mechanisms of glaucoma. We previously reported glaucoma in DBA/2J mice caused by recessive alleles at two loci, isa and ipd, that cause iris stromal atrophy and iris pigment dispersion, respectively. A approach for identifying modifier genes is to study the effects of specific mutations in different mouse strains. When the phenotypic effect of a mutation is modified upon its introduction into a new strain, crosses between the parental strains can be used to identify modifier genes. The purpose of this study was to determine if the effects of the DBA/2J derived isa and ipd loci are modified in strain AKXD-28/Ty. RESULTS: AKXD-28/Ty mice develop glaucoma characterized by intraocular pressure elevation, retinal ganglion loss, and optic nerve excavation. In AKXD-28/Ty, isa causes an iris stromal atrophy phenotype as in DBA/2J. However, the iris pigment dispersion phenotype associated with ipd in DBA/2J does not occur in AKXD-28/Ty. Additionally, a greater severity and speed of retinal and optic nerve damage following intraocular pressure elevation in AKXD-28/Ty compared to DBA/2J mice suggests that AKXD-28/Ty is more susceptible to pressure-induced cell death. CONCLUSIONS: The consequences of the ipd and isa mutations are modified in the AKXD-28/Ty background. These strains provide a resource for the identification of modifier genes that modulate pigment dispersion and susceptibility to pressure-induced cell death.

Animals↗

A screen for dominant modifiers of the irreC-rst cell death phenotype in the developing Drosophila retina.

Programmed cell death (PCD) in the Drosophila retina requires activity of the irregular chiasmC-roughest (irreC-rst) gene. Loss-of-function mutations in irreC-rst block PCD during retinal development and lead to a rough eye phenotype in the adult. To identify genes that interact with irreC-rst and may be involved in PCD, we conducted a genetic screen for dominant enhancers and suppressors of the adult rough eye phenotype. We screened 150,000 mutagenized flies and recovered 170 dominant modifiers that localized primarily to the second and third chromosomes. At least two allelic groups correspond to previously identified death regulators, Delta and dRas1. Examination of retinae from homozygous viable mutants indicated two major phenotypic classes. One class exhibited pleiotropic defects while the other class exhibited defects specific to the cell population that normally undergoes PCD.

Alleles↗

Role of epistatic (modifier) genes in the modulation of the phenotypic diversity of sickle cell anemia.

Sickle hemoglobin is the product of one mutated gene, but the disease phenotype is the product of many genes. Polymorphism among the genes responsible for the pleotropic effects can be epistatic (or modifier) genes contributing to interindividual variation that characterizes sickle cell anemia patients. Modulation in the hemoglobin F levels is associated with the beta-globin gene cluster haplotypes and to gender and chromosomal sites different from chromosome 11 influencing the severity of the disease. Coexistence of alpha thalassemia with sickle cell disease produces hematologic and clinical consequences that are beneficial in some complications but deleterious in others. There is little if any modulation of the phenotype of sickle cell anemia by coexistence of G6PD deficiency. Mutations that favor blood coagulation or thrombosis may influence the phenotype of the disease. Improved understanding of the influence of genes involved in modulating the complex pathophysiology of sickle cell disease may allow prediction of the phenotype of sickle cell patients and aid in management decisions.

Anemia, Sickle Cell↗

Dominant modifiers of the polyhomeotic extra-sex-combs phenotype induced by marked P element insertional mutagenesis in Drosophila.

Members of the Polycomb group (Pc-G) and trithorax group (trx-G) of genes, as well as the enhancers of trx-G and Pc-G (ETP), function together to maintain segment identity during Drosophila development. In order to obtain new marked P mutations in these genes, we screened for dominant modifiers of the extra-sex-combs phenotype displayed by males mutant for the polyhomeotic (ph) gene, a member of the Pc-G group. Five P(lacW) insertions in four different genes were found to stably suppress ph: two are allelic to trithorax, one is the first allele specific to the Minute(2)21C gene, and the remaining two define new trx-G genes, toutatis (tou) in 48A and taranis (tara) in 89B10-13. tou is predicted to encode a 3109 amino acid sequence protein (TOU), which contains a TAM DNA-binding domain, a WAKZ motif, two PHD zinc fingers and a C-terminal bromodomain, and as such is likely to be involved in regulation of chromatin structure as a subunit of a novel chromatin remodelling complex. In a previous study, we found that insertion of a P(ph) transposable element containing ph regulatory sequences creates a high frequency of mutations modifying ph homeotic phenotypes. One such insertion enhanced the ph phenotype and we show that it is a new allele of UbcD1/eff, a gene encoding a ubiquitin-conjugating enzyme that is involved in telomere association and potentially in chromatin remodelling.

Animals↗

Role of the 2 adenine (g.11293_11294insAA) insertion polymorphism in the 3' untranslated region of the factor VII (FVII) gene: molecular characterization of a patient with severe FVII deficiency.

Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients.

3' Untranslated Regions↗

Apoptosis during bone-like tissue development in vitro.

We present evidence of cell death by apoptosis during the development of bone-like tissue formation in vitro. Fetal rat calvaria-derived osteoblasts differentiate in vitro, progressing through three stages of maturation: a proliferation period, a matrix maturation period when growth is downregulated and expression of the bone cell phenotype is induced, and a third mineralization stage marked by the expression of bone-specific genes. Here we show for the first time that cells differentiating to the mature bone cell phenotype undergo programmed cell death and express genes regulating apoptosis. Culture conditions that modify expression of the osteoblast phenotype simultaneously modify the incidence of apoptosis. Cell death by apoptosis is directly demonstrated by visualization of degraded DNA into oligonucleosomal fragments after gel electrophoresis. Bcl-XL, an inhibitor of apoptosis, and Bax, which can accelerate apoptosis, are expressed at maximal levels 24 h after initial isolation of the cells and again after day 25 in heavily mineralized bone tissue nodules. Bcl-2 is expressed in a reciprocal manner to its related gene product Bcl-XL with the highest levels observed during the early post-proliferative stages of osteoblast maturation. Expression of p53, c-fos, and the interferon regulatory factors IRF-1 and IRF-2, but not cdc2 or cdk, were also induced in mineralized bone nodules. The upregulation of Msx-2 in association with apoptosis is consistent with its in vivo expression during embryogenesis in areas that will undergo programmed cell death. We propose that cell death by apoptosis is a fundamental component of osteoblast differentiation that contributes to maintaining tissue organization.

Animals↗

C1q deficiency and autoimmunity: the effects of genetic background on disease expression.

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 x C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp(+/+) strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp(+/+) animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp(+/+) mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp(+/+) strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.

Animals↗