Cystathionine beta-synthase null homocystinuric mice fail to exhibit altered hemostasis or lowering of plasma homocysteine in response to betaine treatment.

Cystathionine beta-synthase (CBS) deficient homocystinuria is an inherited metabolic defect that if untreated typically results in mental retardation, thromboembolism and a range of connective tissue disturbances. A knockout mouse model has previously been used to investigate pathogenic mechanisms in classical homocystinuria (Watanabe et al., PNAS 92 (1995) 1585-1589). This mouse model exhibits a semi-lethal phenotype and the majority of mice do not survive the early neonatal period. We report here that the birth incidence of cbs (-/-) mice produced from heterozygous crosses is non-Mendelian and not significantly improved by treatment with either the Hcy lowering compound betaine or the cysteine donor N-acetylcysteine. Betaine treatment did improve survival of cbs (-/-) mice and restored fertility to female cbs (-/-) mice but did so without significantly lowering Hcy levels. Surviving cbs (-/-) mice failed to show any alteration in coagulation parameters compared to wild-type controls. Moribund cbs (-/-) mice exhibited severe liver injury and hepatic fibrosis while surviving cbs (-/-) mice although less severely affected, still exhibited a level of severe liver injury that is not found in the human disease. The hepatopathy observed in this model may offer an explanation for the failure of cbs (-/-) mice to respond to betaine or exhibit a hypercoagulative phenotype. We conclude that although this model provides useful data on the biochemical sequelae of classical homocystinuria, it does not successfully recapitulate a number of important features of the human disease and its use for studying mechanisms in homocystinuria should be treated with caution as the hepatopathy produces changes which could influence the results.

A novel transgenic mouse model of CBS-deficient homocystinuria does not incur hepatic steatosis or fibrosis and exhibits a hypercoagulative phenotype that is ameliorated by betaine treatment.

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated "human only" (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.

Mouse model of fragile X syndrome: behavioral and hormonal response to stressors.

Fragile X syndrome, a form of mental retardation caused by inadequate levels of fragile X mental retardation protein (FMRP), is characterized by extreme sensitivity to sensory stimuli and increased behavioral and hormonal reactivity to stressors. Fmr1 knockout mice lack FMRP and exhibit abnormal responses to auditory stimuli. This study sought to determine whether Fmr1 knockout mice on an F1 hybrid background are normal in their response to footshock. Knockout mice were also examined for signs of hyperexcitation across an extended trial range, and serum corticosterone levels were evaluated in response to various stressors. The ability to acquire conditioned taste aversion was also assessed. Knockout mice exhibited no impairment in associative aversive learning or memory, since they successfully expressed conditioned taste aversion. Footshock-sensitivity, freezing behavior, and corticosterone response to various stressors did not differ between knockout and wild-type mice. However, knockout mice exhibited significantly increased responses during the extended test. The knockout mice's increased responsiveness to footshock in the extended test may be an indication of increased vulnerability to stress or enhanced emotional reactivity.

Impaired sustained attention and error-induced stereotypy in the aged Ts65Dn mouse: a mouse model of Down syndrome and Alzheimer's disease.

This study compared performance of 15- to 17-month-old Ts65Dn mice to that of littermate controls on an automated sustained attention task in which the location, onset time, and duration of brief visual cues varied unpredictably. Ts65Dn mice committed more omission errors than controls, particularly on trials with the briefest cues. Videotape data revealed that the trisomic mice attended less than controls during the period before cue presentation and engaged in stereotypic jumping and grooming immediately after making an error. These findings reveal that Ts65Dn mice are impaired in sustaining attention and exhibit heightened reactivity to committing an error, and support the validity of this mouse model for studying Down syndrome and Alzheimer's disease. The attention task, coupled with the videotape analyses of task performance, provides a useful paradigm for studying attention and reactivity to errors in mice.

Automated analysis of foot-shock sensitivity and concurrent freezing behavior in mice.

Foot-shock is used in a variety of behavioral tasks either as a motivational stimulus, a way to characterize different rodents, or to test various pharmacological agents for their antinociceptive or analgesic effects. All these procedures need to assess foot-shock sensitivity either to rule out possible differences (when the shock is used as a motivational stimulus) or to use the differences to compare animals or treatments. In addition, many of the procedures that utilize foot-shock as a motivational stimulus evaluate freezing as an index of anxiety or fear. In the present study, data obtained by an automated computer system was compared with data obtained by human observers to validate the automated system for examining foot-shock sensitivity in mice. The different computer measures obtained for foot-shock sensitivity exhibited high correlations with human scoring at shock levels as low as 0.2 mA. The computer controlled analysis provided a non-subjective, quantifiable measurement of the foot-shock-induced response as well as freezing behavior. Automated data collection is an improvement over the methods of human visual observation in that the data collection is more standardized, efficient and consistent.

Reactivity to object and spatial novelty is normal in older Ts65Dn mice that model Down syndrome and Alzheimer's disease.

Ts65Dn mice, a model for Down syndrome and Alzheimer's disease, have a spontaneous age-related reduction of cholinergic markers in medial septal neurons, hippocampal abnormalities, and an age-related learning deficit in a task that requires an intact hippocampus. Others have shown that when normal rodents explored an open field with objects, they detected the displacement of some of the familiar objects within the arena (spatial novelty) and the presence of a new object (object novelty); whereas rodents with hippocampal, fornix, or neonatal selective basal forebrain cholinergic lesions were impaired in detecting spatial, but not object, novelty. In this study, both control and Ts65Dn mice responded to both the spatial and object changes. This unexpected finding could have several explanations. One may be related to recent studies that suggest that only rats with neonatal, but not adult, basal forebrain cholinergic 192 IgG-saporin lesions are impaired in reacting to spatial novelty.

Alterations in the auditory startle response in Fmr1 targeted mutant mouse models of fragile X syndrome.

Fragile X syndrome results from inadequate production of the fragile X mental retardation protein (FMRP). Mice with a mutation targeted to the Fmr1 gene lack FMRP and thus are a valuable animal model for studying the behavioral and neural phenotype of this human disorder. Mice of two genetic backgrounds containing the Fmr1 mutation, C57BL/6J (C57-KO) and an F1 hybrid (C57BL/6J mutant x FVB/NJ; F1-KO) did not differ from control mice in behavior in the elevated plus maze or the open field. Both the C57-KO and F1-KO mice exhibited greater startle responses than normal mice to low intensity (80 dB) white noise bursts and decreased responses to high intensity (120 dB) white noise bursts. These behavioral alterations appear to be specific to the Fmr1 mutation since they are present on both genetic backgrounds. Furthermore, the mice lacking FMRP resemble individuals with fragile X syndrome in their increased sensitivity to low intensity auditory stimuli. These findings should prove useful in determining how the absence of FMRP alters the brain and behavior, and in testing potential treatments for fragile X syndrome.

The Colorado mental retardation and developmental disabilities research center.

The Colorado MRRC was one of the original MRRCs funded and has maintained its focus on genetic and nutritional causes of mental retardation and developmental disabilities. Significant discoveries of the center have included a number of metabolic disorders, including glutaric academia types I and II, electron transport flavoprotein (ETF) deficiency, ETF dehydrogenase deficiency, glycerol kinase deficiency, sphingolipidoses, genetic linkages in dyslexia, phonological deficits in dyslexia, and the importance of the trace mineral Zn in early development. Current research at the center is supported by program of projects grants on inborn errors of metabolism, Down syndrome (DS), autism, and dyslexia.

Estrogen restores cognition and cholinergic phenotype in an animal model of Down syndrome.

Estrogen maintains normal function of basal forebrain (BF) cholinergic neurons and estrogen replacement therapy (ERT) has therefore been proposed as a therapy for Alzheimer's disease (AD). We provide evidence to support this hypothesis in an animal model of Down syndrome (DS), a chromosome 16 segmental trisomy (Ts65Dn) mouse. These mice develop cholinergic degeneration similar to young adults with DS and AD patients. ERT has not been tested in women with DS, even though they are more likely than normosomic women to develop early menopause and AD. Female Ts65Dn and normosomic mice (11-15 months) received a subcutaneous estrogen pellet or a sham operation. After 60 days, estrogen treatment improved learning of a T-maze task and normalized behavior in the Ts65Dn mice in reversal learning of the task, a measure of cognitive flexibility. Stereological evaluation of choline acetyltransferase (ChAT) immunopositive BF neurons showed that estrogen increased cell size and total number of cholinergic neurons in the medial septum of Ts65Dn mice. In addition, estrogen increased NGF protein levels in the BF of trisomic mice. These findings support the emerging hypothesis that estrogen may play a protective role during neurodegeneration and cognitive decline, particularly in cholinergic BF neuronal systems underlying cognition. The findings also indicate that estrogen may act, at least partially, via endogenous growth factors. Collectively, the data suggest that ERT may be a viable therapeutic approach for women with DS coupled with dementia.

Elevated plus maze behavior, auditory startle response, and shock sensitivity in predisease and in early stage autoimmune disease MRL/lpr mice.

Behaviors indicative of anxiety have been suggested to emerge with the onset of autoimmune disease in MRL/MpJ-Fas(lpr) (MRL/lpr) mice. This study extends the behavioral characterization of MRL/lpr and congenic MRL/MpJ+/+ (MRL/+) mice using the elevated plus maze (EPM), acoustic startle response, and foot-shock sensitivity tasks. In the elevated plus maze, predisease MRL/lpr mice exhibited less anxiety while MRL/lpr mice in the early stage of autoimmunity did not differ from age-matched control MRL/+ mice. MRL/lpr mice exhibited lower startle responses compared to MRL/+ mice. Similarly, predisease MRL/lpr mice were less reactive to various foot-shock levels than MRL/+ mice. Both the MRL/lpr and the MRL/+ strains exhibited startle habituation deficits, implicating the background MRL strain in the impairment in this process. These data do not support the hypothesis that increased anxiety is apparent with the emergence of autoimmune disease in MRL/lpr mice; however, anxiety may appear as the disease advances.

Building protein interaction maps for Down's syndrome.

Now that the complete sequences for human chromosome 21 and the orthologous mouse genomic regions are known, reasonably complete, conserved, protein-coding gene catalogues are also available. The central issue now facing Down's syndrome researchers is the correlation of increased expression of specific, normal, chromosome 21 genes with the development of specific deficits in learning and memory. Because of the number of candidate genes involved, the number of alternative splice variants of individual genes and the number of pathways in which these genes function, a pathway analysis approach will be critical to success. Here, three examples, both gene specific and pathway related, that would benefit from pathway analysis are discussed: (1) the potential roles of eight chromosome 21 proteins in RNA processing pathways; (2) the chromosome 21 protein intersectin 1 and its domain composition, alternative splicing, protein interactions and functions; and (3) the interactions of ten chromosome 21 proteins with components of the mitogen-activated protein kinase and the calcineurin signalling pathways. A productive approach to developing gene-phenotype correlations in Down's syndrome will make use of known and predicted functions and interactions of chromosome 21 genes to predict pathways that may be perturbed by their increased levels of expression. Investigations may then be targeted in animal models to specific interactions, intermediate steps or end-points of such pathways and the downstream - perhaps amplified - consequences of gene dosage directly assessed. Once pathway perturbations have been identified, the potential for rational design of therapeutics becomes practical.

Biochemical, pathologic and behavioral analysis of a mouse model of glutaric acidemia type I.

Glutaric acidemia type I (GA-I) is an autosomal recessive disorder of amino acid metabolism resulting from a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients accumulate glutaric acid (GA) and 3-OH glutaric acid (3-OHGA) in their blood, urine and CSF. Clinically, GA-I is characterized by macrocephaly, progressive dystonia and dyskinesia. Degeneration of the caudate and putamen of the basal ganglia, widening of the Sylvian fissures, fronto-temporal atrophy and severe spongiform change in the white matter are also commonly observed. In this report we describe the phenotype of a mouse model of GA-I generated via targeted deletion of the Gcdh gene in embryonic stem cells. The Gcdh-/- mice have a biochemical phenotype very similar to human GA-I patients, including elevations of GA and 3-OHGA at levels similar to those seen in GA-I patients. The affected mice have a mild motor deficit but do not develop the progressive dystonia seen in human patients. Pathologically, the Gcdh-/- mice have a diffuse spongiform myelinopathy similar to that seen in GA-I patients. However, unlike in human patients, there is no evidence of neuron loss or astrogliosis in the striatum. Subjecting the Gcdh-/- mice to a metabolic stress, which often precipitates an encephalopathic crisis and the development of dystonia in GA-I patients, failed to have any neurologic effect on the mice. We hypothesize that the lack of similarity in regards to the neurologic phenotype and striatal pathology of GA-I patients, as compared with the Gcdh-/- mice, is due to intrinsic differences between the striata of mice and men.

Vasoactive intestinal peptide in the brain of a mouse model for Down syndrome.

The most common genetic cause of mental retardation is Down syndrome, trisomy of chromosome 21, which is accompanied by small stature, developmental delays, and mental retardation. In the Ts65Dn segmental trisomy mouse model of Down syndrome, the section of mouse chromosome 16 most homologous to human chromosome 21 is trisomic. This model exhibits aspects of Down syndrome including growth restriction, delay in achieving developmental milestones, and cognitive dysfunction. Recent data link vasoactive intestinal peptide malfunction with developmental delays and cognitive deficits. Blockage of vasoactive intestinal peptide during rodent development results in growth and developmental delays, neuronal dystrophy, and, in adults, cognitive dysfunction. Also, vasoactive intestinal peptide is elevated in the blood of newborn children with autism and Down syndrome. In the current experiments, vasoactive intestinal peptide binding sites were significantly increased in several brain areas of the segmental trisomy mouse, including the olfactory bulb, hippocampus, cortex, caudate/putamen, and cerebellum, compared with wild-type littermates. In situ hybridization for VIP mRNA revealed significantly more dense vasoactive intestinal peptide mRNA in the hippocampus, cortex, raphe nuclei, and vestibular nuclei in the segmental trisomy mouse compared with wild-type littermates. In the segmental trisomy mouse cortex and hippocampus, over three times as many vasoactive intestinal peptide-immunopositive cells were visible than in wild-type mouse cortex. These abnormalities in vasoactive intestinal peptide parameters in the segmental trisomy model of Down syndrome suggest that vasoactive intestinal peptide may have a role in the neuropathology of Down-like cognitive dysfunction.