ABSTRACT
While there is now substantial evidence that 5-HT(6) antagonism leads to significantly improved cognitive ability, the mechanism(s) and/or pathway(s) involved are poorly understood. We have evaluated the consequence of chronic administration of the 5-HT(6) receptor antagonists SB-271046 and SB-399885 on neural cell adhesion molecule polysialylation state (NCAM PSA), a neuroplastic mechanism necessary for memory consolidation. Quantitative analysis of NCAM PSA immunopositive neurons in the dentate gyrus of drug-treated animals revealed a dose-dependent increase in polysialylated cell frequency following treatment with both SB-271046 and SB-399885. These effects could not be attributed to increased neurogenesis, as no difference in the rate of bromodeoxyuridine incorporation was apparent between the control and drug-treated groups. A substantial increase in the frequency of polysialylated cells in layer II of the entorhinal and perirhinal cortices was also observed, brain regions not previously associated with neurogenesis. Chronic treatment with SB-271046 or SB-399885 also significantly increased the activation of dentate polysialylation that is specific to learning. This effect does not occur with other cognition-enhancing drugs, such as tacrine, and this action potentially differentiates 5-HT(6) receptor antagonism as an unique neuroplastic mechanism for cognitive processes which may slow or reverse age/neurodegenerative related memory deficits.
Subject(s)
Dentate Gyrus/drug effects , Hippocampus/drug effects , Neural Cell Adhesion Molecules/pharmacology , Neurons/metabolism , Piperazines/pharmacology , Receptors, Serotonin/drug effects , Serotonin Antagonists/pharmacology , Sialic Acids/pharmacology , Sulfonamides/pharmacology , Thiophenes/pharmacology , Animals , Antimetabolites , Bromodeoxyuridine , Cell Proliferation/drug effects , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Dentate Gyrus/cytology , Dose-Response Relationship, Drug , Entorhinal Cortex/cytology , Entorhinal Cortex/drug effects , Hippocampus/cytology , Immunohistochemistry , Male , Maze Learning/drug effects , Neurons/drug effects , Rats , Rats, WistarSubject(s)
Biomedical Research , Pharmaceutical Preparations , Drug Approval , Drug Industry , Europe , Humans , PatientsABSTRACT
The mechanisms that regulate bone mass are important in a variety of complex diseases such as osteopenia and osteoporosis. Regulation of bone mass is a polygenic trait and is also influenced by various environmental and lifestyle factors, making analysis of the genetic basis difficult. As an effort toward identifying novel genes involved in regulation of bone mass, N-ethyl-N-nitrosourea (ENU) mutagenesis in mice has been utilized. Here we describe a mouse mutant termed Yoda that was identified in an ENU mutagenesis screen for dominantly acting mutations. Mice heterozygous for the Yoda mutation exhibit craniofacial abnormalities: shortened snouts, wider skulls, and deformed nasal bones, underlined by altered morphology of frontonasal sutures and failure of interfrontal suture to close. A major feature of the mutant is reduced bone mineral density. Homozygosity for the mutation results in embryonic lethality. Positional cloning of the locus identified a missense mutation in a highly conserved region of the ankyrin repeat domain 11 gene (Ankrd11). This gene has not been previously associated with bone metabolism and, thus, identifies a novel genetic regulator of bone homeostasis.
Subject(s)
Abnormalities, Multiple/genetics , Bone Diseases, Metabolic/genetics , Craniofacial Abnormalities/genetics , DNA-Binding Proteins/physiology , Kyphosis/genetics , Mice, Mutant Strains/genetics , Mutation, Missense , Amino Acid Sequence , Amino Acid Substitution , Animals , Bone Density/genetics , Conserved Sequence , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Ethylnitrosourea , Female , Genes, Lethal , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Mutant Strains/embryology , Molecular Sequence Data , Mutagenesis , Phenotype , Point Mutation , Repressor Proteins , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
UNLABELLED: Deactivating mutations in the TNSALP gene cause HPP. Akp2(-/-) mice model severe infantile HPP, but there is no model for the relatively mild adult form. Here we report on mice with an induced mutation in Akp2 that affects splicing. The phenotype of homozygotes mirror aspects of the adult form of HPP. INTRODUCTION: Hypophosphatasia (HPP) is a clinically varied skeletal disorder resulting from deficiency of tissue nonspecific alkaline phosphatase (TNSALP). Mice lacking Akp2 model infantile HPP characterized by skeletal hypomineralization, impaired growth, seizures, and perinatal mortality. No animal model exists to study the less severe forms of the disease that typically present in later life. MATERIALS AND METHODS: N-ethyl-N-nitrosourea (ENU) mutagenesis was used to generate mouse models of human disease. A mouse with low plasma alkaline phosphatase (ALP) activity was identified by our clinical chemistry screen. Its offspring were used for inheritance studies and subjected to biochemical, histological, and radiological phenotyping. DNA was extracted for mapping and osteoblasts harvested for functional studies. RESULTS: We showed semidominant inheritance of the low ALP phenotype and mapped the underlying point mutation to Akp2. Affected offspring bear the splice site mutation 862 + 5G>A-a hypomorphic allele named Akp2(Hpp). The same mutation has been reported in a patient. Akp2(Hpp/+) mice have approximately 50% of normal plasma ALP but display no other biochemical or skeletal abnormalities. Unlike Akp2(-/-) mice, Akp2(Hpp/Hpp) mice have normal initial skeletal development and growth, a normal lifespan and do not have seizures. TNSALP is low but detectable in Akp2(Hpp/Hpp) plasma. Osteoblasts display approximately 10% of normal ALP activity and reduced intracellular inorganic phosphate levels, yet are capable of normal mineralization in vitro. TNSALP substrates are significantly elevated in urine (inorganic pyrophosphate and phosphoethanolamine) and plasma (pyridoxal 5'-phosphate), whereas plasma inorganic pyrophosphate levels are normal. Akp2(Hpp/Hpp) mice develop late-onset skeletal disease, notably defective endochondral ossification and bone mineralization that leads to arthropathies of knees and shoulders. CONCLUSIONS: Akp2(Hpp/Hpp) mice mirror a number of clinical features of the human adult form of HPP. These mice provide for the first time an animal model of late onset HPP that will be valuable in future mechanistic studies and for the evaluation of therapies such as those aimed at HPP.
Subject(s)
Alkaline Phosphatase/genetics , Disease Models, Animal , Genes, Dominant , Hypophosphatasia/genetics , Mutation , RNA Splicing , Animals , Base Sequence , DNA, Complementary , Mice , Microscopy, Electron, Scanning , Molecular Sequence Data , PhenotypeABSTRACT
Otitis media (OM), inflammation of the middle ear, is the most common cause of hearing impairment and surgery in children. Recurrent and chronic forms of OM are known to have a strong genetic component, but nothing is known of the underlying genes involved in the human population. We have previously identified a novel semi-dominant mouse mutant, Jeff, in which the heterozygotes develop chronic suppurative OM (Hardisty, R.E., Erven, A., Logan, K., Morse, S., Guionaud, S., Sancho-Oliver, S., Hunter, A.J., Brown, S.D. and Steel, K.P. (2003) The deaf mouse mutant Jeff (Jf) is a single gene model of otitis media. J. Assoc. Res. Otolaryngol., 4, 130-138.) and represent a model for chronic forms of OM in humans. We demonstrate here that Jeff carries a mutation in an F-box gene, Fbxo11. Fbxo11 is expressed in epithelial cells of the middle ears from late embryonic stages through to day 13 of postnatal life. In contrast to Jeff heterozygotes, Jeff homozygotes show cleft palate, facial clefting and perinatal lethality. We have also isolated and characterized an additional hypomorphic mutant allele, Mutt. Mutt heterozygotes do not develop OM but Mutt homozygotes also show facial clefting and cleft palate abnormalities. FBXO11 is one of the first molecules to be identified, contributing to the genetic aetiology of OM. In addition, the recessive effects of mutant alleles of Fbxo11 identify the gene as an important candidate for cleft palate studies in the human population.
Subject(s)
F-Box Proteins/genetics , F-Box Proteins/metabolism , Mutation/genetics , Otitis Media/genetics , Otitis Media/pathology , Proteins/genetics , Proteins/metabolism , Alleles , Amino Acid Sequence , Animals , F-Box Proteins/chemistry , Homozygote , Mice , Molecular Sequence Data , Otitis Media/metabolism , PhenotypeABSTRACT
Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM.
Subject(s)
DNA-Binding Proteins/genetics , Genetic Predisposition to Disease/genetics , Mutation/genetics , Otitis Media/genetics , Proto-Oncogenes/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Chromosome Mapping , Cloning, Molecular , DNA Mutational Analysis , DNA-Binding Proteins/chemistry , Ear, Middle/cytology , Ear, Middle/pathology , Flow Cytometry , Granulocytes/immunology , Lung/cytology , Lung/pathology , MDS1 and EVI1 Complex Locus Protein , Male , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Molecular Sequence Data , Nose/cytology , Nose/pathology , Otitis Media/immunology , Phenotype , Specific Pathogen-Free Organisms , Transcription Factors/chemistryABSTRACT
RATIONALE: Neuromedin-U (NmU) is an agonist at NMU1R and NMU2R. The brain distribution of NmU and its receptors, in particular NMU2R, suggests widespread central roles for NmU. In agreement, centrally administered NmU affects feeding behaviour, energy expenditure and pituitary output. Further central nervous system (CNS) roles for NmU warrant investigation. OBJECTIVES: To investigate the CNS role of NmU by mapping NMU1R and NMU2R mRNA and measuring the behavioural, endocrine, neurochemical and c-fos response to intracerebroventricular (i.c.v.) NmU. METHODS: Binding affinity and functional potency of rat NmU was determined at human NMU1R and NMU2R. Expression of NMU1R and NMU2R mRNA in rat and human tissue was determined using semi-quantitative reverse-transcription polymerase chain reaction. In in-vivo studies, NmU was administered i.c.v. to male Sprague-Dawley rats, and changes in grooming, motor activity and pre-pulse inhibition (PPI) were assessed. In further studies, plasma endocrine hormones, [DOPAC + HVA]/[dopamine] and [5-HIAA]/[5-HT] ratios and levels of Fos-like immunoreactivity (FLI) were measured 20 min post-NmU (i.c.v.). RESULTS: NmU bound to NMU1R ( K(I), 0.11+/-0.02 nM) and NMU2R ( K(I), 0.21+/-0.05 nM) with equal affinity and was equally active at NMU1R (EC(50), 1.25+/-0.05 nM) and NMU2R (EC(50), 1.10+/-0.20 nM) in a functional assay. NMU2R mRNA expression was found at the highest levels in the CNS regions of both rat and human tissues. NMU1R mRNA expression was restricted to the periphery of both species with the exception of the rat amygdala. NmU caused a marked increase in grooming and motor activity but did not affect PPI. Further, NmU decreased plasma prolactin but did not affect levels of corticosterone, luteinising hormone or thyroid stimulating hormone. NmU elevated levels of 5-HT in the frontal cortex and hypothalamus, with decreased levels of its metabolites in the hippocampus and hypothalamus, but did not affect dopamine function. NmU markedly increased FLI in the nucleus accumbens, frontal cortex and central amygdala. CONCLUSIONS: These data provide further evidence for widespread roles for NmU and its receptors in the brain.
Subject(s)
Central Nervous System/drug effects , Central Nervous System/metabolism , Membrane Proteins/agonists , Membrane Proteins/metabolism , Neuropeptides/administration & dosage , Receptors, Neurotransmitter/agonists , Receptors, Neurotransmitter/metabolism , Animals , Cell Line , Dose-Response Relationship, Drug , Humans , Injections, Intraventricular , Rats , Rats, Sprague-Dawley , SwineABSTRACT
The purpose of the present set of studies was to develop a new primate model of focal ischemia with reperfusion for long-term functional assessment in the common marmoset. Initially, the cerebral vascular anatomy of the marmoset was interrogated by Araldite-cast and ink-perfusion methods to determine the feasibility of an intravascular surgical approach. The methods showed that the internal carotid artery was highly tortuous in its passage, precluding the development of an extracranial method of inducing temporary middle cerebral artery occlusion in the marmoset. A pilot dose-response study investigated an intracranial approach of topically applying endothelin-1 (ET-1) to the M2 portion of the middle cerebral artery in a small sample of marmosets for up to 6 hours (n = 2 or 3 per group). Dose-dependent reductions in middle cerebral artery vessel caliber followed by gradual reperfusion were inversely related to increases in corrected lesion volume after ET-1 treatment, relative to vehicle control application. Finally, the functional consequences of ET-1-induced lesions to the M2 vascular territory were assessed up to 24 hours after surgery using the optimal dose established in the pilot study (2.5 nmol/25 microL). ET-1-treated marmosets (n = 4) showed marked contralateral motor deficits in grip strength and retrieval of food rewards and contralateral sensory/motor neglect towards tactile stimulation, relative to their ipsilateral side and vehicle-treated marmosets (n = 4). Strong correlations were shown between contralateral impairments and histopathologic parameters, which revealed unilateral putamen and cortical damage to the middle cerebral artery territory. No deficits were shown on general mobility, and self-care was promptly resumed in ET-1 marmosets after surgery. These results show that this novel model of ischemia with reperfusion in the marmoset has the potential to assess long-term function and to gauge the efficacy of novel therapeutic strategies targeted for clinical stroke.
Subject(s)
Endothelin-1/pharmacology , Infarction, Middle Cerebral Artery/chemically induced , Infarction, Middle Cerebral Artery/pathology , Reperfusion Injury/pathology , Stroke/physiopathology , Animals , Behavior, Animal/drug effects , Brain/pathology , Callithrix , Conditioning, Operant/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Epoxy Resins , Female , Forelimb/physiology , Hand Strength/physiology , Hindlimb/physiology , Infarction, Middle Cerebral Artery/psychology , Male , Perfusion , Phthalic Anhydrides , Physical Stimulation , Pilot Projects , Reflex/physiology , Reperfusion Injury/psychology , Reward , Stroke/psychology , Vocalization, Animal/physiologyABSTRACT
Three mutant mice with pigmentation phenotypes were recovered from a genomewide random mouse chemical mutagenesis study. White toes (Whto; MGI:1861986), Belly spot and white toes (Bswt; MGI:2152776) and Dark footpads 2 (Dfp2; MGI:1861991) were identified following visual inspection of progeny from a male exposed to the point mutagen ethylnitrosourea (ENU). In order to rapidly localize the causative mutations, genome-wide linkage scans were performed on pooled DNA samples from backcross animals for each mutant line. Whto was mapped to proximal mouse chromosome (Mmu) 7 between Cen (the centromere) and D7Mit112 (8.0 cM from the centromere), Bswt was mapped to centric Mmul between D1Mit214 (32.1 cM) and D1Mit480 (32.8 cM) and Dfp2 was mapped to proximalMmu4 between Cen and D4Mit18 (5.2 cM). Whto, Bswt and Dfp2 may provide novel starting points in furthering the elucidation of genetic and biochemical pathways relevant to pigmentation and associated biological processes.
ABSTRACT
Otitis media is the most common cause of hearing impairment in children and is primarily characterized by inflammation of the middle ear mucosa. Yet nothing is known of the underlying genetic pathways predisposing to otitis media in the human population. Increasingly, large-scale mouse mutagenesis programs have undertaken systematic and genome-wide efforts to recover large numbers of novel mutations affecting a diverse array of phenotypic areas involved with genetic disease including deafness. As part of the UK mutagenesis program, we have identified a novel deaf mouse mutant, Jeff (Jf). Jeff maps to the distal region of mouse chromosome 17 and presents with fluid and pus in the middle ear cavity. Jeff mutants are 21% smaller than wild-type littermates, have a mild craniofacial abnormality, and have elevated hearing thresholds. Middle ear epithelia of Jeff mice show evidence of a chronic proliferative otitis media. The Jeff mutant should prove valuable in elucidating the underlying genetic pathways predisposing to otitis media.
Subject(s)
Deafness/genetics , Disease Models, Animal , Mice, Mutant Strains/genetics , Otitis Media with Effusion/genetics , Proteins/genetics , Animals , Auditory Threshold , Body Constitution , Chromosome Mapping , Chronic Disease , Craniofacial Abnormalities/genetics , Deafness/physiopathology , Humans , Mice , Otitis Media with Effusion/pathology , Otitis Media with Effusion/physiopathology , SuppurationABSTRACT
The robotic mouse is an autosomal dominant mutant that arose from a large-scale chemical mutagenesis program. It has a jerky, ataxic gait and develops adult-onset Purkinje cell loss in the cerebellum in a striking region-specific pattern, as well as cataracts. Genetic and physical mapping of the disease locus led to the identification of a missense mutation in a highly conserved region of Af4, a putative transcription factor that has been previously implicated in leukemogenesis. We demonstrate that Af4 is specifically expressed in Purkinje cells, and we hypothesize that the expression of mutant Af4 leads to neurodegeneration. This function was not identified through knock-out studies, highlighting the power of phenotype-driven mutagenesis in the mouse to identify new pathways involved in neurological disease.
Subject(s)
Cataract/genetics , Cerebellar Ataxia/genetics , Cerebellum/pathology , DNA-Binding Proteins/genetics , Nuclear Proteins/genetics , Purkinje Cells/pathology , Amino Acid Sequence , Animals , Antigens, CD/biosynthesis , Cell Count , Cerebellar Ataxia/pathology , Conserved Sequence , DNA-Binding Proteins/biosynthesis , Disease Models, Animal , Disease Progression , Flow Cytometry , Genes, Dominant , Mice , Mice, Neurologic Mutants , Molecular Sequence Data , Mutagenesis , Nuclear Proteins/biosynthesis , Organ Specificity/genetics , Physical Chromosome Mapping , Point Mutation , Purkinje Cells/metabolism , Sequence Homology, Amino Acid , Thymus Gland/metabolism , Thymus Gland/pathologyABSTRACT
The semi-dominantly inherited mouse mutation pardon (Pdo) was isolated due to the lack of a Preyer reflex (ear flick) in response to sound from a large-scale N -ethyl- N -nitrosourea (ENU) mutagenesis programme. Dissection of the middle ear revealed malformations in all three ossicles, rendering the ossicular chain incomplete. Hair cell counts in the apical turn of the organ of Corti revealed a significant 22.7% increase in the number of outer hair cells. Raised compound action potential thresholds in Pdo/+ mutants suggested a combined sensorineural/conductive hearing loss. We show that a missense mutation in the homeobox gene Emx2 is responsible for these defects, identifying a new function for this gene in the development of specific structures in the ear.
Subject(s)
Cochlea/pathology , Ear, Middle/pathology , Hearing Loss, Conductive/pathology , Hearing Loss, Sensorineural/pathology , Homeodomain Proteins/genetics , Action Potentials/genetics , Animals , Auditory Threshold/physiology , Cochlea/abnormalities , Cochlea/physiopathology , Ear Ossicles/abnormalities , Ear Ossicles/pathology , Ear Ossicles/physiopathology , Ear, Middle/abnormalities , Ear, Middle/physiopathology , Female , Gene Expression Regulation, Developmental/genetics , Hair Cells, Auditory, Outer/abnormalities , Hair Cells, Auditory, Outer/pathology , Hair Cells, Auditory, Outer/physiopathology , Hearing Loss, Conductive/genetics , Hearing Loss, Conductive/physiopathology , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/physiopathology , Homeodomain Proteins/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Mutant Strains , Microscopy, Electron, Scanning , Molecular Sequence Data , Mutation, Missense/genetics , Sequence Homology, Amino Acid , Transcription FactorsABSTRACT
Expression levels of mRNA are commonly measured as a ratio of test to reference gene. The assumption is that reference genes such as beta-actin or cyclophilin are unaffected by treatment and act as steady-state controls. TaqMan real-time RT-PCR was used to test these assumptions in a rat model of cerebral ischaemia (tMCAO). Following measurement of 24 genes, we show that reference genes in this animal model fail the criteria for steady-state controls. Neuronal loss, glial proliferation and an influx of leukocytes into the lesioned brain result in major disturbance to cell populations. The mRNA for reference genes, as for test genes, reflects these changes. Specific mRNA levels vary according to the choice of reference gene to which they are normalised. In the process of resolving reference gene issues, mRNA increases were discovered for leukaemia inhibitory factor, nestin and galanin in rat brain hemispheres affected by ischaemia. Results are reported for a further 21 genes and mathematical and statistical methods are described that allow in this study fraction-fold changes in mRNA to be detected.
Subject(s)
Brain Ischemia/genetics , Gene Expression Regulation , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Animals , Data Interpretation, Statistical , Disease Models, Animal , Infarction, Middle Cerebral Artery , Male , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Reference Standards , Reverse Transcriptase Polymerase Chain Reaction/standardsABSTRACT
We have identified and characterized a new peripheral myelin protein 22 (Pmp22) mouse mutant. The mutation results in a serine to threonine amino acid substitution at residue 72, which is a hot spot for mutation in human PMP22, leading to the peripheral neuropathy Dejerine-Sottas syndrome. We have previously described two other Pmp22 mutants, providing an allelic series for gene function analysis. Pmp22 mutations generally lead to abnormal intracellular trafficking of Pmp22, and we show that each mutant protein in the allelic series has a unique pattern of intracellular localization in transfected cell lines. The mutant protein from the less severely affected mutants occurs in large aggregates, while the mutant protein from the most severely affected mutant occurs in a diffuse perinuclear pattern that largely colocalizes with wild-type protein. This suggests that large Pmp22 aggregates may be protective in this form of peripheral neuropathy.
Subject(s)
Mice, Neurologic Mutants/genetics , Myelin Proteins/genetics , Peripheral Nervous System Diseases/genetics , Peripheral Nervous System Diseases/metabolism , Alleles , Animals , COS Cells , Dimerization , Gene Expression/genetics , Genotype , HeLa Cells , Humans , Mice , Microscopy, Electron , Myelin Sheath/metabolism , Myelin Sheath/pathology , Myelin Sheath/ultrastructure , Peripheral Nervous System Diseases/pathology , Phenotype , Sciatic Nerve/metabolism , Sciatic Nerve/pathology , Sciatic Nerve/ultrastructure , TransfectionABSTRACT
We have carried out a genome-wide screen for novel N-ethyl-N-nitrosourea-induced mutations that give rise to eye and vision abnormalities in the mouse and have identified 25 inherited phenotypes that affect all parts of the eye. A combination of genetic mapping, complementation and molecular analysis revealed that 14 of these are mutations in genes previously identified to play a role in eye pathophysiology, namely Pax6, Mitf, Egfr and Pde6b. Many of the others are located in genomic regions lacking candidate genes and these define new loci. Four of the mutants display a similar phenotype of dilated pupils but do not appear to be allelic, and at least two of these are embryonic lethal when homozygous. This collection of eye mutations will be valuable for understanding gene function, for dissecting protein function and as models of human eye disease.