Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation with neuronal vulnerability in Huntington's disease.

Immunohistochemistry and single-cell RT-PCR were used to characterize the localization of huntingtin and/or its mRNA in the major types of striatal neurons and in corticostriatal projection neurons in rats. Single-label immunohistochemical studies revealed that striatum contains scattered large neurons rich in huntingtin and more numerous medium-sized neurons moderate in huntingtin. Double-label immunohistochemical studies showed that the large huntingtin-rich striatal neurons include nearly all cholinergic interneurons and some parvalbuminergic interneurons. Somatostatinergic striatal interneurons, which are medium in size, rarely contained huntingtin. Calbindin immunolabeling showed that the vast majority of the medium-sized striatal neurons that contain huntingtin are projection neurons, but only approximately 65% of calbindin-labeled projection neurons (localized to the matrix compartment of striatum) were labeled for huntingtin. Calbindin-containing projection neurons of the matrix compartment and calbindin-negative projection neurons of the striatal patch compartment contained huntingtin with comparable frequency. Single-cell RT-PCR confirmed that striatal cholinergic interneurons contain huntingtin, but only approximately 65% of projection neurons contained detectable huntingtin message. The finding that huntingtin is not consistently found in striatal projection neurons [which die in Huntington's disease (HD)] but is abundant in striatal cholinergic interneurons (which survive in Huntington's disease) suggests that the mutation in huntingtin that causes HD may not directly kill neurons. In contrast to the heterogeneous expression of huntingtin in the different striatal neuron types, we found all corticostriatal neurons to be rich in huntingtin protein and mRNA. One possibility raised by our findings is that the HD mutation may render corticostriatal neurons destructive rather than render striatal neurons vulnerable.

Identification and cloning of a brain autoantigen in neuro-behavioral SLE.

In murine models of SLE, particular patterns of abnormalities of social interaction and memory collectively known as neurobehavioral dysfunction (NBD) correlate with the occurrence of brain reactive autoantibodies. Study of the immunopathogenic effects of these antibodies has been limited by the absence of isolated autoantibodies and antigens. In order to identify the molecular targets, we isolated autoantibodies highly specific for brain plasma membranes from MRL/lpr mice. After immunoscreening a brain expression library with these brain specific autoantibodies, we identified a single cDNA clone of unique sequence and relevant anatomic distribution. Transcript for this cDNA is wide spread among mammalian species but appears to be present only in the brain. Addition features, suggesting this cDNA is pertinent for further study include (1) the expressed protein, called lupus brain antigen 1, reacts with the screening immunoglobulins as well as immunoglobulins from other strains of murine neuro-SLE not used to screen the library, but not with immunoglobulins from normal mice, (2) the transcript distribution within the brain is similar to immunochemical localization of binding of the spontaneous autoantibodies and (3) the localization of transcript within the brain, in the hippocampus, hypothalamus an cingulate gyrus, corresponds to anticipated anatomical regions of clinical dysfunction. Further, the transcript is a large, potentially structural molecule of unique sequence. Antibodies to this molecule may mediate changes in behavior either by direct interactions with the cognate antigen or by indirect influences through neuro-endocrine axes.

NMDA receptor overstimulation triggers a prolonged wave of immediate early gene expression: relationship to excitotoxicity.

Exposure of the rodent striatum to quinolinic acid (QA, N-methyl-D-aspartate receptor agonist) induces immediate early gene (IEG; c-fos, c-jun, jun-B, zif/268) expression that may extend 12-24 h after injection. In order to determine the specificity of the prolonged IEG response to the QA injection, the temporal pattern of c-fos mRNA expression was examined during the first 4 h after administration of saline or QA (40 micrograms). As early as 30 min after intrastriatal injection, both saline and QA increased c-fos mRNA levels. In the saline group, this increase in IEG expression was only transient and returned to baseline by 1 h. In contrast, c-fos mRNA levels within QA-injected animals continued to rise significantly at 1 and 4 h. In a second experiment, rats received 4 ng to 40-micrograms injections of QA followed by sacrifice at 6 h to determine if increasing QA doses caused the appearance of the prolonged IEG response phase. The prolonged IEG response was evident at 6 h only in animal groups that received higher dose ranges (4-40 micrograms) of QA. A final experiment was undertaken to determine if blockage of NMDA receptor stimulation would also inhibit the prolonged IEG response at 6 h in relationship to neuronal sparing evidenced at 24 h post-QA injection. The NMDA receptor antagonist, MK-801, blocked the prolonged IEG response at 6 h following QA (40 micrograms) injection while also preventing striatal neuropeptide mRNA decline by 24 h. Delaying the MK-801 administration for 1-2 h post-QA injection revealed that the intensity of the prolonged IEG mRNA response may be predictive of neuronal demise within the QA lesion site. These results suggest that prolonged IEG expression is associated with QA excitotoxicity of the rodent striatum and subsequent neuronal degeneration.

Preprotachykinin and preproenkephalin mRNA expression within striatal subregions in response to altered serotonin transmission.

The effects of lowered serotonin (5-hydroxytryptamine; 5-HT) neurotransmission on preprotachykinin (PPT) and preproenkephalin (PPE) mRNA levels were examined in subregions of the striatum. Adult male rats were treated systemically with para-chlorophenylalanine (pCPA; 350 mg/kg single i.p. injection) which reduced forebrain 5-HT amounts to approximately 20% of saline-injected controls at 24 and 48 h. As measured by Northern analysis, PPT and PPE mRNA levels were elevated 50% and 160% respectively in the anterior ventromedial striatum (region included nucleus accumbens). PPT mRNA levels were raised 90% in posterior striatum (at the level of the globus pallidus) by 48 h post-pCPA injection. To determine if increased PPT and PPE mRNA levels represented a transient response to brief 5-HT inhibition, additional experiments were performed to provide continual suppression of 5-HT within the striatum. First, rats received daily intraperitoneal injections of saline or the 5-HT1A receptor agonist, 8-OH-DPAT (1 mg/kg), for 7 days to reduce 5-HT release from raphestriatal terminals. In a parallel experiment, the serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT, 5 micrograms), was stereotaxically injected into the striatum as a means to permanently remove 5-HT terminals. Although levels of each mRNA species were differentially sensitive to 5,7-DHT or 8-OH-DPAT, PPT and PPE mRNAs were lowered between 30-55% within the anterior dorsolateral and ventromedial striatum. Although these results support previous studies suggesting an overall positive regulatory role of serotonin on striatal tachykinin biosynthesis, PPT and PPE gene regulation in certain striatal subregions may by differentially sensitive to lowered 5-HT neurotransmission. This suggestion is supported by observations that acute systemic stimulation of 5-HT2A/C receptors with DOI (7 mg/kg single i.p. injection) raised PPT and PPE mRNA levels within anterior dorsolateral (30-60%) and posterior (100-200%) striata, but not within the anterior ventromedial striatum.

The identification of a functional nuclear localization signal in the Huntington disease protein.

Positional cloning has shown that the Huntington disease (HD) mutation is an expanded trinucleotide repeat in the IT15 gene. Although this mutation clearly produces the HD phenotype, the function of the Huntington disease protein remains undefined. One recent immunocytochemical study suggested that the IT15 protein preferentially localizes to the nucleus of affected neuronal cells. If this result is accurate, it could link the biochemical function of this protein to nuclear activities such as gene regulation. To examine the nuclear transport of the Huntington disease protein, we searched for basic peptide motifs that could produce nuclear localization. One peptide (RRKGKEK) was identified that is highly homologous to a consensus nuclear localization signal. When fused to the cytoplasmic reporter protein, beta-galactosidase, nuclear localization was observed in stably transformed human cell lines. In a complementary study, an anti-peptide polyclonal antibody, raised against a sequence adjacent to the putative nuclear localization sequence, detected the IT15 protein in the nucleus of human cells. These results extend and confirm the previous localization studies and identify an IT15 peptide motif that can function for nuclear localization.

Immediate early gene activation during the initial phases of the excitotoxic cascade.

Direct brain injections of the N-methyl-D-aspartate receptor agonist quinolinic acid (QA) trigger an excitotoxic cascade characterized by rapid neuronal death and glial/immune cell activation. The present study compared the timing of immediate early gene (IEG; c-fos, c-jun, jun-B, and zif/268) induction with the response of neuronal transcripts during the first 24 hr of a QA lesion within the rodent striatum. Following QA exposure, IEG mRNA induction periods extended from 30 min to 24 hr. Several characteristics of this prolonged transcriptional response suggest that separate cell populations (neuronal vs. glial) originate individual IEG phases during the first day of the lesion. The first IEG phase was rapid and peaked at 60 min. This initial IEG phase, likely neuronal in origin, was dominated by robust increases in the expression of c-fos, jun-B, and zif/268 mRNAs in contrast to small increases in c-jun expression. A second, delayed IEG phase was initiated after the first hour and extended to 24 hr. This IEG phase was more intense and continued beyond the period of neuronal survival as detected by the loss of neurotransmitter-specific mRNAs (preprotachykinin, preproenkephalin, and glutamic acid decarboxylase). During this phase, c-jun mRNA levels coordinately increased with c-fos. Interestingly, the transcriptional peak of the delayed IEG phase occurred between 4 and 12 hr, the time which corresponded to the rapid decline of neuronal transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

Timing the excitotoxic induction of heat shock protein 70 transcription.

The time course of heat-shock protein 70 (HSP70) transcriptional induction was compared with neuronal- and non-neuronal-specific mRNAs following intrastriatal quinolinate (QA) injection. Within one hour of QA exposure, immediate-early gene (IEG; c-fos) activation preceded slight increases in glutamic acid decaroxylase (GAD) mRNA levels. However, glial fibrillary acid protein (GFAP) and HSP70 mRNA levels remained constant. After one hour, HSP70 mRNA levels surged 6 fold during a delayed transcriptional phase that was induced between 4-12 hours. This phase was characterized by massive increases in c-fos and GFAP mRNAs while GAD transcripts fell drastically suggestive of neuronal death. Therefore HSP70 genes may play an important role in glial/immune activation following rapid excitotoxic damage by direct injections of quinolinate.

Progress towards cloning the cystic fibrosis gene.

Genetic linkage analysis with polymorphic DNA markers (restriction fragment length polymorphisms: RFLPS) has allowed the assignment of the cystic fibrosis (CF) locus to the long arm of chromosome 7, within the region of band q31. Two of these markers, MET and D7S8, are tightly linked to the disease locus. Although recent data suggest that they are located on opposite sides of CF, the two can be separated by as much as 5 centimorgans. To obtain a better description of the CF locus and, eventually, to identify the affected gene, additional DNA markers are required to connect MET and D7S8, physically. We have screened the flow-sorted chromosome-7-specific library and thus far isolated 28 new probes from the 7q31 region by DNA hybridization analysis that uses a series of somatic cell hybrids containing various portions of human chromosome 7. Together with the previously identified markers, MET, D7S8, D7S13 and D7S16, these new markers should provide a fine genetic and physical map for the chromosomal region surrounding CF. DNA segments can then be sequentially cloned by chromosome walking from points closest to the CF locus and examined for genes that are preferentially expressed in tissues known to be affected in the disease.

cDNA cloning and chromosomal localization (4q11-13) of a gene for statherin, a regulator of calcium in saliva.

On the basis of the known amino acid sequence of statherin, a human salivary protein, mixed synthetic oligonucleotides were synthesized and used to screen a cDNA library constructed from human parotid-gland mRNA. A cDNA clone coding for statherin was isolated from this library and has been completely sequenced. The cDNA represents a full-length (or nearly full-length) copy of an approximately 640-bp statherin mRNA. Statherin appears to be coded by a single-copy gene that maps to chromosome 4q11-4q13 when somatic-cell hybrids are used.

Rapid RFLP screening procedure identifies new polymorphisms at albumin and alcohol dehydrogenase loci.

A rapid screening procedure for restriction fragment length polymorphisms (RFLPs) is reported. DNA from ten individuals is pooled and compared to DNA isolated from a cell line containing a single chromosome 4. This single chromosome-containing line is an obligate hemizygote for chromosome 4 RFLPs so that only one band corresponding to a single allele will appear on a Southern blot. In the pooled DNA sample lane bands corresponding to both alleles will be seen. The technique allows for efficient detection of RFLPs with easier use of large numbers of enzymes. It provides estimates of allele frequencies and disequilibrium. New RFLPs for albumin and alcohol dehydrogenase detected with this technique are described.

Chromosomal localization of human gene for histidyl-tRNA synthetase: clustering of genes encoding aminoacyl-tRNA synthetases on human chromosome 5.

The gene encoding histidyl-tRNA synthetase was localized to human chromosome 5 using human-Chinese hamster ovary cell hybrids. Seven of the functionally related genes encoding the 20 different aminoacyl-tRNA synthetases have not been mapped in humans and four are located on a single chromosome, number 5, which represents less than 7% of the total genome. Thus, there appears to be significant clustering of this group of genes which may reflect their evolutionary relatedness or common factors involved in regulating their expression.

A cell hybrid and recombinant DNA library that facilitate identification of polymorphic loci in the vicinity of the Huntington disease gene.

Somatic cell hybrids were selected that retain a derivative chromosome 5 from an individual in which the p15.1-pter segment of chromosome 5 is replaced with the p15.1-pter segment of chromosome 4. Hybrids that retain this derivative chromosome exclusively were found to be positive for G8, a DNA marker closely linked to the Huntington disease gene on chromosome 4p. From one such hybrid, a segregant was isolated that had deleted the entire q arm of the derivative chromosome but retained the p arm intact as its only detectable human DNA. A complete recombinant DNA library was prepared from this cell line, and the inserts in approximately 1/3 of the recombinant phage with human DNA were shown to be derived from 4pter-4p15.1, which represents only approximately 1% of the total human genome. The cell hybrid and DNA library represent a rapid and efficient means to identify and isolate many polymorphic DNA markers close to and flanking the Huntington disease gene.

Genetic counterselective procedure to isolate interspecific cell hybrids containing single human chromosomes: construction of cell hybrids and recombinant DNA libraries specific for human chromosomes 3 and 4.

Counterselection against genes on human chromosome 5 was applied to interspecific human-Chinese hamster cell hybrids which retained this and one additional human chromosome in order to generate cell hybrids retaining single, nonselected human chromosomes. Using this procedure, stable cell hybrids which retain human chromosome 3 exclusively or human chromosome 4 exclusively were isolated. Complete recombinant genomic DNA libraries were prepared from each hybrid using the lambda cloning vector EMBL-4. These libraries represent sources of human DNA fragments derived specifically from chromosomes 3 and 4, respectively. Low-copy or unique human DNA fragments isolated from both libraries were analyzed to confirm their chromosomal origin and to determine the complexity of their hybridization patterns to total human DNA. These single human chromosome libraries represent a means to efficiently saturate chromosomes 3 and 4 with informative, polymorphic genetic markers. DNA fragments from the chromosome 4 library will be particularly useful in identifying additional genetic markers close to the Huntington's disease gene. The same genetic counterselective procedure can be utilized to derive several additional cell hybrids with single human chromosomes.

Deletion mapping of human chromosome 5 using chromosome-specific DNA probes.

A complete genomic DNA library was prepared from a Chinese hamster-human cell hybrid that contains human chromosome 5 as its only human DNA. Unique or low-copy DNA fragments, isolated form recombinant bacteriophage that contained human DNA inserts, were regionally mapped on chromosome 5 using Southern blot analysis of genomic DNA from a series of hybrid cell lines that were selected as having deletions of various portions of 5q. The chromosome 5-specific DNA library, together with a genetic selective procedure allowing the isolation of hybrid cell lines with deletions of virtually any portion of 5q, will provide a means to construct very accurate physical and recombinational maps of this human chromosome. This system represents an excellent opportunity to examine very precisely the relationship between physical and genetic distances for many loci along the length of this autosome.

Molecular approach to analyzing the human 5p deletion syndrome, cri du chat.

DNA unique or low-copy fragments were isolated from a genomic DNA library specific for the short (p) arm of human chromosome 5. These chromosome 5p-specific DNA fragments were used to analyze, by Southern blot experiments, somatic cell hybrids that retained either a normal chromosome 5 homolog or a homolog with a partial deletion of 5p, which was derived from either of two persons with the common human deletion syndrome, cri du chat or 5p- syndrome. In these studies, two classes of DNA fragments were identified, those located outside the region deleted in the persons with cri du chat and those located within the deleted region. This latter class of DNA probes will help to define, at the molecular level, a region of 5p that is critical in producing the phenotype associated with the cri du chat syndrome.

Transformation-defective mutant of adenovirus type 5 containing a single altered E1a mRNA species.

A mutant of adenovirus type 5 containing an octanucleotide insert in region E1a of the viral genome was constructed. The insert was present in only one (13s) of the three overlapping mRNA's synthesized from this region. The insert was within the sequences removed by RNA splicing during the production of the other two nRNA's. The insertion resulted in a shift in the translational reading frame of the 13s mRNA and the probable premature termination of translation. The mutant was defective for viral DNA replication in HeLa cells and the transformation of rat embryo and baby rat kidney cells, indicating that a product encoded by the 13s nRNA is required for these two processes. Other early regions of the genome were expressed in HeLa cells infected by this mutant although in some cases the expression was decreased as compared with wild-type-infected cells.