CAG repeat lengths > or =335 attenuate the phenotype in the R6/2 Huntington's disease transgenic mouse.

With spontaneous elongation of the CAG repeat in the R6/2 transgene to > or =335, resulting in a transgene protein too large for passive entry into nuclei via the nuclear pore, we observed an abrupt increase in lifespan to >20 weeks, compared to the 12 weeks common in R6/2 mice with 150 repeats. In the > or =335 CAG mice, large ubiquitinated aggregates of mutant protein were common in neuronal dendrites and perikaryal cytoplasm, but intranuclear aggregates were small and infrequent. Message and protein for the > or =335 CAG transgene were reduced to one-third that in 150 CAG R6/2 mice. Neurological and neurochemical abnormalities were delayed in onset and less severe than in 150 CAG R6/2 mice. These findings suggest that polyQ length and pathogenicity in Huntington's disease may not be linearly related, and pathogenicity may be less severe with extreme repeats. Both diminished mutant protein and reduced nuclear entry may contribute to phenotype attenuation.

Increased calbindin-D28k immunoreactivity in striatal projection neurons of R6/2 Huntington's disease transgenic mice.

Striatal degeneration in Huntington's disease (HD) is associated with increases in perikaryal calbindin immunolabeling in yet-surviving striatal projection neurons. Since similar increases have also been observed in surviving striatal projection neurons after intrastriatal injection of the excitotoxin quinolinic acid, the increased calbindin in HD striatum has been interpreted to suggest an excitotoxic process in HD. We used immunolabeling to assess if calbindin is elevated in striatal projection neurons of R6/2 HD transgenic mice. These mice bear exon 1 of the human huntingtin gene with 144 CAG repeats and show some of the neuropathological signs (e.g., neuronal intranuclear inclusions) and clinical traits (e.g., wasting prior to early death) of HD. We found an increased frequency of calbindin-immunoreactive neuronal perikarya in the striatum of 6- and 12-week-old R6/2 mice compared to wild-type controls. This increase was most notable in the normally calbindin-poor dorsolateral striatum. We found no significant changes in the total area of striatum occupied by the calbindin-negative striosomes and no consistent changes in striatal calbindin mRNA. The increase in calbindin in R6/2 striatal neurons was thus limited to the matrix compartment, and it may be triggered by increased Ca2+ entry due to the demonstrated heightened NMDA sensitivity of these neurons. The data further support the similarity of R6/2 mice to HD, and are consistent with the occurrence of an excitotoxic process in striatum in both.

Identification of surface-membrane P-type ATPases resembling fungal K(+)- and Na(+)-ATPases, in Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani.

Genomic DNA fragments encoding nine, novel, P-type ATPases in trypanosomatid organisms were amplified in PCR, using degenerate oligonucleotide primers that recognize the ATP-binding and -phosphorylation sites present in all P-type ATPases. Subsequent phylogenetic analysis, based on the presence of conserved motifs in predicted peptide sequences for six Trypanosoma brucei, T. cruzi or Leishmania donovani PCR fragments, identified calcium-, proton- and phospholipid-translocating ATPases. DNA fragments that predict proteins homologous to the fungal, type-IID, P-type, ATPase pumps that transport Na(+) or K(+) ions were also present in T. brucei (TBCA1; 1022 nucleotides representing 340 amino acids), T. cruzi (TCNA1; 1022 nucleotides representing 340 amino acids) and L. donovani (LDCA1; 1031 nucleotides representing 343 amino acids). Southern blots showed that the Na(+)-ATPases were each present as a single-copy gene. The LDCA1 fragment was used to clone the complete LDCA1 gene from an L. donovani genomic-DNA library. The LDCA1 gene encodes a protein, of 1047 amino acids, with a predicted molecular mass of 115,501 Da. The results of analyses based on northern blots and the rapid amplification of cDNA ends (RACE) indicated that LDCA1 was expressed in promastigotes and amastigotes from axenic cultures and in animal-derived amastigotes. TBCA1 was expressed, as a 5.0-kb transcript, in procyclic culture stages and bloodstream trypomastigotes, with the 5.0-kb message up-regulated six-fold in the trypomastigote stage. Western blots probed with an antibody to the partial TBCA1 peptide identified a 150-kDa protein that was detected, by immunofluorescence, on the surface membrane of procyclic T. brucei.

Neurons lacking huntingtin differentially colonize brain and survive in chimeric mice.

To determine whether neurons lacking huntingtin can participate in development and survive in postnatal brain, we used two approaches in an effort to create mice consisting of wild-type cells and cells without huntingtin. In one approach, chimeras were created by aggregating the 4-8 cell embryos from matings of Hdh (+/-) mice with wild-type 4-8 cell embryos. No chimeric offspring that possessed homozygous Hdh (-/-) cells were obtained thereby, although statistical considerations suggest that such chimeras should have been created. By contrast, Hdh (-/-) ES cells injected into blastocysts yielded offspring that were born and in adulthood were found to have Hdh (-/-) neurons throughout brain. The Hdh (-/-) cells were, however, 5-10 times more common in hypothalamus, midbrain, and hindbrain than in telencephalon and thalamus. Chimeric animals tended to be smaller than wild-type littermates, and chimeric mice rich in Hdh (-/-) cells tended to show motor abnormalities. Nonetheless, no brain malformations or pathologies were evident. The apparent failure of aggregation chimeras possessing Hdh (-/-) cells to survive to birth is likely attributable to the previously demonstrated critical role of huntingtin in extraembryonic membranes. That Hdh (-/-) cells in chimeric mice created by blastocyst injection are under-represented in adult telencephalon and thalamus implies a role for huntingtin in the development of these regions, whereas the neurological dysfunction in brains enriched in Hdh (-/-) cells suggests a role for huntingtin in adult brain. Nonetheless, the lengthy survival of Hdh (-/-) cells in adult chimeric mice indicates that individual neurons in many brain regions do not require huntingtin to participate in normal brain development and to survive.

Abnormalities in the functioning of adipocytes from R6/2 mice that are transgenic for the Huntington's disease mutation.

In an effort to characterize the basis of abnormalities in body weight regulation (i.e. wasting) in Huntington's disease (HD), we examined adipocytes in a transgenic model of HD, the R6/2 mouse. These mice typically show severe wasting beginning at approximately 12 weeks of age and die between 12 and 15 weeks. Despite an overall growth retardation compared with wild-type littermates, we observed an enhanced accumulation of body fat at 8-9 weeks of age in R6/2 mice fed laboratory chow or a synthetic high fat, high sugar diet. The obesity was not accompanied by symptoms associated with diabetes, as there were no abnormalities in serum glucose, serum insulin or the ability of insulin to stimulate glucose metabolism in epididymal adipose tissue. As expected, the obesity in the high fat, high sugar-fed R6/2 mice was accompanied by increased serum leptin. The ability of insulin to stimulate leptin release from isolated epididymal adipose tissue was also enhanced in R6/2 mice. In contrast, the ability of isoproterenol to inhibit leptin release was reduced in adipose tissue from R6/2 mice, as was the lipolytic effect of isoproterenol. These data suggest that the obesity observed at 8-9 weeks in R6/2 mice may stem from a defect in fat breakdown by adipocytes.

Transient global ischemia in rats yields striatal projection neuron and interneuron loss resembling that in Huntington's disease.

The various types of striatal projection neurons and interneurons show a differential pattern of loss in Huntington's disease (HD). Since striatal injury has been suggested to involve similar mechanisms in transient global brain ischemia and HD, we examined the possibility that the patterns of survival for striatal neurons after transient global ischemic damage to the striatum in rats resemble that in HD. The perikarya of specific types of striatal interneurons were identified by histochemical or immunohistochemical labeling while projection neuron abundance was assessed by cresyl violet staining. Projectionneuron survival was assessed by neurotransmitter immunolabeling of their efferent fibers in striatal target areas. The relative survival of neuron types was determined quantitatively within the region of ischemic damage, and the degree of fiber loss in striatal target areas was quantified by computer-assisted image analysis. We found that NADPHd(+) and cholinergic interneurons were largely unaffected, even in the striatal area of maximal damage. Parvalbumin interneurons, however, were as vulnerable as projection neurons. Among immunolabeled striatal projection systems, striatoentopeduncular fibers survived global ischemia better than did striatopallidal or striatonigral fibers. The order of vulnerability observed in this study among the striatal projection systems, and the resistance to damage shown by NADPHd(+) and cholinergic interneurons, is similar to that reported in HD. The high vulnerability of projection neurons and parvalbumin interneurons to global ischemia also resembles that seen in HD. Our results thus indicate that global ischemic damage to striatum in rat closely mimics HD in its neuronal selectivity, which supports the notion that the mechanisms of injury may be similar in both.

A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections.

The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10-50 mM sodium citrate solution (pH 8.5-9.0) preheated to and maintained at 80 degrees C in a water-bath, followed by 30 min incubation in 0.3-3% nonfat dry milk to reduce nonspecfic staining. This method is highly effective for both 40 microm free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure.