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C A Ross

Publications and source records attributed to C A Ross.

At least 73 records · Page 4Linked to original sources
PubMed1999

Preparation of human cDNas encoding expanded polyglutamine repeats.

At least eight neurodegenerative diseases result from expansions of polyglutamine tracts encoded by CAG trinucleotide repeats. Although polyglutamine diseases typically have onset after age 50 in humans, these diseases can be modeled in animals and in cell culture by using highly expanded repeats to accelerate the pathogenesis. Unfortunately, current methods for preparing recombinant constructs with large glutamine tracts either alter the coding region adjacent to the repeat or yield highly unstable pure CAG repeats. We have developed a technique for expanding repeats that results in a more stable mix of CAG and CAA glutamine codons. We expect this technique to allow rapid preparation of highly expand repeats suitable for stable animal and cell culture models for any of the polyglutamine repeat diseases.

DNA, Complementary↗
PubMed1999

Heligmosomoides polygyrus and Trypanosoma congolense infections in mice: effect of immunisation by abbreviated larval infection.

Concurrent African trypanosome and gastrointestinal helminth infections are prevalent in sub-humid savannah where they are endemic. However, acquired resistance in animals varies with their responder status and exposure. As a guide to study in the definitive hosts, the effects of Trypanosoma congolense infection on the development and maintenance of homologous Heligmosomoides polygyrus resistance were investigated in outbred TO mice. These mice were immunised by abbreviation of larval infection. Immune or naive mice were either infected with 500 infective larvae (L3) of H. polygyrus and/or 10(4) bloodstream forms of T. congolense or were not infected. The outcome of infection was monitored by routine parasitological and immunological techniques for 30 days after the day of the T. congolense infection. Significantly more immune mice concurrently infected with both parasites survived than did immune mice in which H. polygyrus was superimposed on a 10-day-old T. congolense infection. Although all the mice in this latter group died before the end of the experiment, larval immunisation prolonged their survival, relative to similarly treated naive mice. The antibody titres to H. polygyrus in the sera of immune mice challenged with H. polygyrus alone were significantly higher than those of immune mice concurrently infected with both parasites but the levels of protection obtained were comparable. It is concluded that T. congolense may not completely block the strong acquired resistance induced by abbreviated H. polygyrus larval infection in TO mice but is capable of interfering with protective responses, especially if the trypanosome infection occurs prior to H. polygyrus challenge infection.

Administration, Oral↗
PubMed1999

Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.

Animals↗
PubMed1999

Rapid mass spectrometric analysis for ascorbate and related organic acids in small volumes of plasma for use in pediatric subjects.

A gas chromatographic-mass spectrometric isotope dilution method was developed for analysis of ascorbate on 10 microl samples of plasma. This assay was reproducible (standard deviation of less than 4%) and gave values for plasma ascorbate content within 8% of our previously published gas chromatographic-mass spectrometric method. Non-specific sample preparation allowed other analytes to be determined on the same sample by adjusting data acquisition parameters and adding the appropriate internal standard. Analysis on 28 subjects fell within the expected range for plasma ascorbate 68+/-29 microm (11.9+/-5.0 microg/ml) and established a normal range for plasma threonate of 28.1+/-2.4 microm (3.8+/-0.4 microg/ml).

Ascorbic Acid↗
PubMed1999

Differential cellular expression of isoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain.

Inositol 1,4,5-trisphosphate receptors (IP3R) are mediators of second messenger-induced intracellular calcium release. Three isoforms are known to be expressed in brain, but their regional distributions and cellular localizations are little known. In order to better understand the roles of IP3 receptor isoforms in brain function, a first step is to define their distributions. We have used affinity-purified antibodies directed against peptides unique to each isoform to determine their sites of expression in rat brain. Type 1 IP3R (IP3R1) is dramatically enriched in Purkinje neurons in cerebellum and neurons in other regions, consistent with previous studies. By contrast, IP3R2 is only detected in glia, whereas IP3R3 is predominantly neuronal, with little detected in glia. IP3R3 is enriched in neuropil, especially in neuronal terminals (which often contain large dense core vesicles) in limbic and basal forebrain regions including olfactory tubercle, central nucleus of the amygdala, and bed nucleus of the stria terminalis. In addition, IP3R1 and IP3R3 have clearly distinct time courses of expression in developing brains. These data suggest separate roles for inositol 1,4,5-trisphosphate receptor isoforms in development, and for glial and neuronal function. The IP3R3 may be involved in regulation of neurotransmitter or neuropeptide release in terminals within specific nuclei of the basal forebrain and limbic system.

Animals↗
PubMed1999

Cleavage of atrophin-1 at caspase site aspartic acid 109 modulates cytotoxicity.

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.

Amino Acid Sequence↗
PubMed1999

Trinucleotide repeat expansion and neuropsychiatric disease.

Trinucleotide, or triplet, repeats consist of 3 nucleotides consecutively repeated (e.g., CCG CCG CCG CCG CCG) within a region of DNA, a not uncommon motif in the genome of humans and other species. In 1991, a new type of genetic mutation was discovered, known as a dynamic or expansion mutation, in which the number of triplets in a repeat increases and the length becomes unstable. During the past decade, nearly 20 diseases-including Huntington disease, 2 forms of the fragile X syndrome, and myotonic dystrophy-caused by trinucleotide repeat expansions have been identified. The unstable nature of the expanded repeat leads to remarkable patterns of inheritance in these diseases, distinctly at odds with traditional notions of mendelian genetics. We review the clinical and genetic features of these disorders, with a particular emphasis on their psychiatric manifestations. We also critically examine the hypothesis that expansion mutations may have an etiologic role in psychiatric diseases such as bipolar disorder, schizophrenia, and autism.

Autistic Disorder↗
PubMed1999

Nuclear targeting of mutant Huntingtin increases toxicity.

Huntington's disease is a neurodegenerative disorder resulting from expansion of the polyglutamine region in huntingtin. Although huntingtin is normally cytoplasmic, in affected brain regions proteolytic fragments of mutant huntingtin containing the polyglutamine repeat form intranuclear inclusions. Here, we examine the contribution of nuclear localization to toxicity by transiently transfecting neuro-2a cells with an N-terminal huntingtin fragment similar in size to that believed to be present in patients. The huntingtin fragment, HD-N63, was targeted either to the cytoplasm with a nuclear export signal (NES) or to the nucleus with a nuclear localization signal (NLS). The NES decreased the number of cells with aggregates in the nucleus while an NLS had the opposite effect. By cotransfecting HD-N63 with GFP as a marker, we observed direct cell loss with constructs containing expanded polyglutamine repeats. Compared to unmodified HD-N63-75Q, adding an NES reduced cell loss by 57% while an NLS increased cell loss by 111%. These results indicate that nuclear localization of mutant huntingtin fragments plays an important role in cell toxicity.

Animals↗
PubMed1999

Immunization of rats reduces nicotine distribution to brain.

The effect of active immunization against nicotine on the initial distribution of nicotine to brain was studied in anesthetized rats. Animals received nicotine 0.03 mg/kg nicotine (equivalent to the nicotine dose absorbed by a human smoking two cigarettes) as a rapid injection in the jugular vein. In control animals, the arterial serum nicotine concentration initially exceeded the venous concentration 4.6-fold, similar to the initial arteriovenous difference produced by cigarette smoking in humans. Animals immunized with the nicotine analog CMUNic maintained this arteriovenous gradient, but with both arterial and venous nicotine concentrations several times higher than in controls. The arterial nicotine concentration was higher in immunized animals even at the first (7.5 s) sampling time. The brain nicotine concentration at 3 min was 36% lower in the immunized animals. The time course of nicotine distribution to brain was studied in a second group of animals. Brain nicotine concentration was reduced in rats immunized with CMUNic over the entire 6-min sampling period immediately following nicotine dosing (mean reduction 38%). A reduction was found at the earliest sampling time (30 s) and was maximal at 1 min (48%). Nicotine protein binding in serum was markedly increased in animals immunized with CMUNic compared to controls (91.2 versus 10.9%), and the unbound nicotine concentration in serum was lower (10.0 versus 13.4 ng/ml). The reduction in brain nicotine concentration correlated with antibody affinity for nicotine, and the percentage of nicotine bound in serum. These data demonstrate that nicotine-specific antibodies produced by active immunization rapidly bind nicotine in arterial blood, reduce the unbound nicotine concentration, and reduce the early distribution of nicotine to brain. Effects were observed using a clinically relevant nicotine dose and route of administration. These data suggest that the use of immunization to modify the behavioral effects of nicotine may be possible.

Animals↗
PubMed1999

Nuclear accumulation of truncated atrophin-1 fragments in a transgenic mouse model of DRPLA.

Dentatorubral and pallidoluysian atrophy (DRPLA) is a member of a family of progressive neurodegenerative diseases caused by polyglutamine repeat expansion. Transgenic mice expressing full-length human atrophin-1 with 65 consecutive glutamines exhibit ataxia, tremors, abnormal movements, seizures, and premature death. These mice accumulate atrophin-1 immunoreactivity and inclusion bodies in the nuclei of multiple populations of neurons. Subcellular fractionation revealed 120 kDa nuclear fragments of mutant atrophin-1, whose abundance increased with age and phenotypic severity. Brains of DRPLA patients contained apparently identical 120 kDa nuclear fragments. By contrast, mice overexpressing atrophin-1 with 26 glutamines were phenotypically normal and did not accumulate the 120 kDa fragments. We conclude that the evolution of neuropathology in DRPLA involves proteolytic processing of mutant atrophin-1 and nuclear accumulation of truncated fragments.

Adolescent↗
PubMed1999

Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues, so abnormalities outside the brain might be expected. Although involvement of nuclei and mitochondria in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains; mitochondria are organelles that regulates apoptotic cell death. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats.

Adolescent↗
PubMed1999

Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions.

Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions. A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein). Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD. It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen. We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP). We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons. Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions.

Amino Acid Sequence↗
PubMed1999

Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin.

Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by the expansion of a glutamine repeat in the N-terminus of the huntingtin protein. To gain insight into the pathogenesis of HD, we generated transgenic mice that express a cDNA encoding an N-terminal fragment (171 amino acids) of huntingtin with 82, 44 or 18 glutamines. Mice expressing relatively low steady-state levels of N171 huntingtin with 82 glutamine repeats (N171-82Q) develop behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait, before dying prematurely. In mice exhibiting these abnormalities, diffuse nuclear labeling, intranuclear inclusions and neuritic aggregates, all immunoreactive with an antibody to the N-terminus (amino acids 1-17) of huntingtin (AP194), were found in multiple populations of neurons. None of these behavioral or pathological phenotypes were seen in mice expressing N171-18Q. These findings are consistent with the idea that N-terminal fragments of huntingtin with a repeat expansion are toxic to neurons, and that N-terminal fragments are prone to form both intranuclear inclusions and neuritic aggregates.

Animals↗