Microtiter plate quantification of mutant and wild-type huntingtin normalized to cell count

Huntington’s disease is caused by a gain-of-function neurotoxic mutation in normally neuroprotective huntingtin. Sensitive assays are required to discriminate mutant huntingtin from wild-type huntingtin. We have developed a normalized 384-plate assay for determination of mutant and wild-type huntingtin. Based on a single pipetting step, the sensitive assay uses two antibody pairs for simultaneous mutant and wild-type huntingtin time-resolved fluorescence resonance energy transfer detection combined with PicoGreen quantification of double-stranded DNA. The assay can be used for discovery of drugs reducing mutant huntingtin over wild-type huntingtin and for assessing the value of huntingtin as a disease progression marker, and it is adaptable to other proteins of interest.     

Microtiter plate quantification of mutant and wild-type huntingtin normalized to cell count

QuikChange is a popular method for site-directed mutagenesis in structural and functional studies of proteins and nucleic acids. However, the standard protocol is often inefficient in producing the desired mutations. Here we present a novel strategy for primer design, central overlapping primers (COP), which employs a pair of bipartite primers of different lengths, with the short primer complementary to the middle region of the long primer. The COP method is efficient and robust in generating approximately 90% mutation rate without supercompetent Escherichia coli cells or laborious screening for positive clones.     

Population stratification may bias analysis of PGC-1α as a modifier of age at Huntington disease motor onset

Huntington’s disease (HD) is an inherited neurodegenerative disorder characterized by motor, cognitive and behavioral disturbances, caused by the expansion of a CAG trinucleotide repeat in the HD gene. The CAG allele size is the major determinant of age at onset (AO) of motor symptoms, although the remaining variance in AO is highly heritable. The rs7665116 SNP in PPARGC1A, encoding the mitochondrial regulator PGC-1α, has been reported to be a significant modifier of AO in three European HD cohorts, perhaps due to affected cases from Italy. We attempted to replicate these findings in a large collection of (1,727) HD patient DNA samples of European origin. In the entire cohort, rs7665116 showed a significant effect in the dominant model (p value?=?0.008) and the additive model (p value?=?0.009). However, when examined by origin, cases of Southern European origin had an increased rs7665116 minor allele frequency (MAF), consistent with this being an ancestry-tagging SNP. The Southern European cases, despite similar mean CAG allele size, had a significantly older mean AO (p?<?0.001), suggesting population-dependent phenotype stratification. When the generalized estimating equations models were adjusted for ancestry, the effect of the rs7665116 genotype on AO decreased dramatically. Our results do not support rs7665116 as a modifier of AO of motor symptoms, as we found evidence for a dramatic effect of phenotypic (AO) and genotypic (MAF) stratification among European cohorts that was not considered in previously reported association studies. A significantly older AO in Southern Europe may reflect population differences in genetic or environmental factors that warrant further investigation.     

Effects of age,replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.     

Context-dependent pollinator behavior: an explanation for patterns of hybridization among three species of Indian paintbrush

In some areas of sympatry, reproductively compatible plant species hybridize, but in other areas of sympatry, they do not and they remain reproductively isolated from one another. Explanations offered to explain patterns of hybridization that vary by population have usually focused on genetic or environmental factors. Instead, we examined whether different community contexts might change pollinator preference and constancy and thus influence the likelihood of hybridization among three Indian paintbrush species (Castilleja miniata, C. rhexifolia, and C. sulphurea). To determine whether visitation was context‐dependent, we observed pollinator behavior in experimental arrays (constructed using flowering stems of the three Indian paintbrush species) in different contexts. Contexts were defined by which Castilleja species occurred in the immediate neighborhood of the arrays. Specifically, we asked, does visitation to particular species in the arrays depend on context? In general, each Castilleja species was preferred when it matched the surrounding community context, as is predicted by optimal foraging theory. More interestingly, pollinator constancy was weakened in the hybrid context (an area where the three species co‐occurred with morphologically intermediate plants), which is likely to increase pollen flow among the species. Reduced pollinator constancy in hybrid zones could set up a positive feedback loop in which more flower diversity is created through hybridization, decreasing pollinator constancy, and leading to more hybridization. This self‐reinforcing mechanism could lead to “hybridization hot spots” and to a patchy distribution of hybrid populations. We expect that this mechanism may be important in other animal‐pollinated plant hybrid zones.     

Mechanisms of copper ion mediated Huntington's disease progression

Huntington's disease (HD) is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgenic mice. Increased brain copper correlated with decreased levels of the copper export protein, amyloid precursor protein. We hypothesized that increased amounts of copper bound to low affinity sites could contribute to pro-oxidant activities and neurodegeneration. We focused on two proteins: huntingtin, because of its centrality to HD, and lactate dehydrogenase (LDH), because of its documented sensitivity to copper, necessity for normoxic brain energy metabolism and evidence for altered lactate metabolism in HD brain. The first 171 amino acids of wild-type huntingtin, and its glutamine expanded mutant form, interacted with copper, but not iron. N171 reduced Cu(2+)in vitro in a 1:1 copper:protein stoichiometry indicating that this fragment is very redox active. Further, copper promoted and metal chelation inhibited aggregation of cell-free huntingtin. We found decreased LDH activity, but not protein, and increased lactate levels in HD transgenic mouse brain. The LDH inhibitor oxamate resulted in neurodegeneration when delivered intra-striatially to healthy mice, indicating that LDH inhibition is relevant to neurodegeneration in HD. Our findings support a role of pro-oxidant copper-protein interactions in HD progression and offer a novel target for pharmacotherapeutics.     

Diverse pre- and post-synaptic expression of m1-m4 muscarinic receptor proteins in neurons and afferents in the rat neostriatum

We have utilized subtype specific antibodies to determine the cellular and subcellular distributions of the muscarinic acetylcholine receptor subtypes that are highly expressed in the rat striatum (m1-m4). Each receptor is expressed in distinct populations of striatal neurons in the relative proportions predicted by their mRNAs. They concentrate at post-synaptic sites and each of the four subtypes are also transported to pre-synaptic sites. m2 appears to be the only presynaptic autoreceptor in the striatum, but it is also localized in non-cholinergic terminals. These distinct pre- and post-synaptic localizations suggest that muscarinic receptor subtype diversity evolved to enable increasingly complex responses to acetylcholine release.