The predominantly HEAT-like motif structure of huntingtin and its association and coincident nuclear entry with dorsal, an NF-kB/Rel/dorsal family transcription factor

BACKGROUND: Huntington's disease (HD) pathogenesis is due to an expanded polyglutamine tract in huntingtin, but the specificity of neuronal loss compared with other polyglutamine disorders also implies a role for the protein's unknown inherent function. Huntingtin is moderately conserved, with 10 HEAT repeats reported in its amino-terminal half. HD orthologues are evident in vertebrates and Drosophila, but not in Saccharomyces cerevisiae, Caenorhabditis elegans or Arabidopsis thaliana, a phylogenetic profile similar to the NF-kB/Rel/dorsal family transcription factors, suggesting a potential functional relationship. RESULTS: We initially tested the potential for a relationship between huntingtin and dorsal by overexpression experiments in Drosophila S2 cells. Drosophila huntingtin complexes via its carboxyl-terminal region with dorsal, and the two enter the nucleus concomitantly, partly in a lipopolysaccharide (LPS)- and Nup88-dependent manner. Similarly, in HeLa cell extracts, human huntingtin co-immunoprecipitates with NF-kB p50 but not with p105. By cross-species comparative analysis, we find that the carboxyl-terminal segment of huntingtin that mediates the association with dorsal possesses numerous HEAT-like sequences related to those in the amino-terminal segment. Thus, Drosophila and vertebrate huntingtins are composed predominantly of 28 to 36 degenerate HEAT-like repeats that span the entire protein. CONCLUSION: Like other HEAT-repeat filled proteins, huntingtin is made up largely of degenerate HEAT-like sequences, suggesting that it may play a scaffolding role in the formation of particular protein-protein complexes. While many proteins have been implicated in complexes with the amino-terminal region of huntingtin, the NF-kB/Rel/dorsal family transcription factors merit further examination as direct or indirect interactors with huntingtin's carboxyl-terminal segment

Huntington's disease: the case for genetic modifiers

For almost three decades, Huntington's disease has been a prototype for the application of genetic strategies to human disease. HD, the Huntington's disease gene, was the first autosomal defect mapped using only DNA markers, a finding in 1983 that helped to spur similar studies in many other disorders and contributed to the concept of the human genome project. The search for the genetic defect itself pioneered many mapping and gene-finding technologies, and culminated in the identification of the HD gene, its mutation and its novel protein product in 1993. Since that time, extensive investigations into the pathogenic mechanism have utilized the knowledge of the disease gene and its defect but, with notable exceptions, have rarely relied for guidance on the genetic findings in human patients to interpret the relevance of findings in non-human model systems. However, the human patient still has much to teach us through a detailed analysis of genotype and phenotype. Such studies have implicated the existence of genetic modifiers - genes whose natural polymorphic variation contributes to altering the development of Huntington's disease symptoms. The search for these modifiers, much as the search for the HD gene did in the past, offers to open new entrées into the process of Huntington's disease pathogenesis by unlocking the biochemical changes that occur many years before diagnosis, and thereby providing validated target proteins and pathways for development of rational therapeutic interventions

Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

BACKGROUND: Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the Hdh(Q111/Q111 )knock-in mouse. RESULTS: We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC(50 )< 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant Hdh(Q111/Q111 )striatal cells. CONCLUSIONS: At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that alter in vitro aggregation is a viable approach for defining potential therapeutic compounds that may act on the deleterious conformational property of full-length mutant huntingtin

Autism Spectrum Disorder Genetics: Diverse Genes with Diverse Clinical Outcomes

The last several years have seen unprecedented advances in deciphering the genetic etiology of autism spectrum disorders (ASDs). Heritability studies have repeatedly affirmed a contribution of genetic factors to the overall disease risk. Technical breakthroughs have enabled the search for these genetic factors via genome-wide surveys of a spectrum of potential sequence variations, from common single-nucleotide polymorphisms to essentially private chromosomal abnormalities. Studies of copy-number variation have identified significant roles for both recurrent and nonrecurrent large dosage imbalances, although they have rarely revealed the individual genes responsible. More recently, discoveries of rare point mutations and characterization of balanced chromosomal abnormalities have pinpointed individual ASD genes of relatively strong effect, including both loci with strong a priori biological relevance and those that would have otherwise been unsuspected as high-priority biological targets. Evidence has also emerged for association with many common variants, each adding a small individual contribution to ASD risk. These findings collectively provide compelling empirical data that the genetic basis of ASD is highly heterogeneous, with hundreds of genes capable of conferring varying degrees of risk, depending on their nature and the predisposing genetic alteration. Moreover, many genes that have been implicated in ASD also appear to be risk factors for related neurodevelopmental disorders, as well as for a spectrum of psychiatric phenotypes. While some ASD genes have evident functional significance, like synaptic proteins such as the SHANKs, neuroligins, and neurexins, as well as fragile x mental retardation–associated proteins, ASD genes have also been discovered that do not present a clear mechanism of specific neurodevelopmental dysfunction, such as regulators of chromatin modification and global gene expression. In their sum, the progress from genetic studies to date has been remarkable and increasingly rapid, but the interactive impact of strong-effect genetic lesions coupled with weak effect common polymorphisms has not yet led to a unified understanding of ASD pathogenesis or explained its highly variable clinical expression. With an increasingly firm genetic foundation, the coming years will hopefully see equally rapid advances in elucidating the functional consequences of ASD genes and their interactions with environmental/experiential factors, supporting the development of rational interventions

Predictive value of hematological and phenotypical parameters on postchemotherapy leukocyte recovery

Background: Grade IV chemotherapy toxicity is defined as absolute neutrophil count &lt;500/μL. The nadir is considered as the lowest neutrophil number following chemotherapy, and generally is not expected before the 7th day from the start of chemotherapy. The usual prophylactic dose of rHu-G-CSF (Filgrastim) is 300 μg/day, starting 24-48 h after chemotherapy until hematological recovery. However, individual patient response is largely variable, so that rHu-G-CSF doses can be different. The aim of this study was to verify if peripheral blood automated flow cytochemistry and flow cytometry analysis may be helpful in predicting the individual response and saving rHu-G-CSF. Methods: During Grade IV neutropenia, blood counts from 30 cancer patients were analyzed daily by ADVIA 120 automated flow cytochemistry analyzer and by Facscalibur flow cytometer till the nadir. "Large unstained cells" (LUCs), myeloperoxidase index (MPXI), blasts, and various cell subpopulations in the peripheral blood were studied. At nadir rHu-G-CSF was started and 81 chemotherapy cycles were analyzed. Cycles were stratified according to their number and to two dose-levels of rHuG-CSF needed to recovery (300-600 vs. 900-1200 μg) and analyzed in relation to mean values of MPXI and mean absolute number of LUCs in the nadir phase. The linear regressions of LUCs % over time in relation to two dose-levels of rHu-G-CSF and uni-multivariate analysis of lymphocyte subpopulations, CD34+ cells, MPXI, and blasts were also performed. Results: In the nadir phase, the increase of MPXI above the upper limit of normality (&gt;10; median 27.7), characterized a slow hematological recovery. MPXI levels were directly related to the cycle number and inversely related to the absolute number of LUCs and CD34 +/CD45+ cells. A faster hematological recovery was associated with a higher LUC increase per day (0.56% vs. 0.25%), higher blast (median 36.7/μL vs. 19.5/μL) and CD34+/CD45+ cell (median 2.2/μL vs. 0.82/μL) counts. Conclusions: Our study showed that some biological indicators such as MPXI, LUCs, blasts, and CD34 +/CD45+ cells may be of clinical relevance in predicting individual hematological response to rHu-G-CSF. Special attention should be paid when nadir MPXI exceeds the upper limit of normality because the hematological recovery may be delayed. © 2009 Clinical Cytometry Society

Unreinforced Masonry Structures‘ Seismic Improvement with F.R.C.M. : the Experience of the Vanvitellian Palazzo Murena of Perugia

The aim of the contribution is to illustrate strengthening design in regard to the conservation of Palazzo Murena in Perugia, designed in the eighteenth century by the prominent Architect Luigi Vanvitelli. Starting from an accurate historical analysis and taking into account experimental campaign and results of numerical analysis, the vulnerable elements of the construction have been highlighted. A local reinforcement intervention is proposed with the aim of retain the historical value of the architectural characteristics; this consists of the application of plaster with Fiber Reinforced Cementitious Matrix, F.R.C.M., to achieve an adequate strength, without adding supplemental weight to the masonry structure and therefore avoiding an increase of the seismic vulnerability. The benefits of the application of F.R.C.M. materials, also with respect to different reinforcement techniques are broaden

The effect of mechanical and geometric uncertainty on perforated CFS bracing members

In the capacity design of cold-formed steel frames with X diagonal bracings, the ratio of overstrength to slenderness is particularly critical. The diagonal elements of these braces may be fabricated with perforations at the brace ends to satisfy design and detailing requirements for capacity protection of frames with concentric X bracings. In the paper, the influence of stochasticity in the geometrical features and mechanical properties on the overall structural response of specific cold-formed steel perforated elements is assessed. The impact of statistical variation in design parameters on the yield strength, ultimate strength, and ductility is evaluated through a Monte Carlo simulation. Variability in member geometric features was determined from current design specifications, while variability in steel mechanical properties was determined via experimental testing. Monte Carlo simulations indicate a slight reduction of yield and ultimate member resistance increasing the number of holes. A normal probability distribution function, with a skewness greater than zero, which increases with a larger number of holes, characterizes both the yield and ultimate strength histogram. The work concludes providing recommendations for designers to promote utilization of perforated braces in seismically-active area

Unbiased Gene Expression Analysis Implicates the huntingtin Polyglutamine Tract in Extra-mitochondrial Energy Metabolism

The Huntington's disease (HD) CAG repeat, encoding a polymorphic glutamine tract in huntingtin, is inversely correlated with cellular energy level, with alleles over ∼37 repeats leading to the loss of striatal neurons. This early HD neuronal specificity can be modeled by respiratory chain inhibitor 3-nitropropionic acid (3-NP) and, like 3-NP, mutant huntingtin has been proposed to directly influence the mitochondrion, via interaction or decreased PGC-1α expression. We have tested this hypothesis by comparing the gene expression changes due to mutant huntingtin accurately expressed in STHdhQ111/Q111 cells with the changes produced by 3-NP treatment of wild-type striatal cells. In general, the HD mutation did not mimic 3-NP, although both produced a state of energy collapse that was mildly alleviated by the PGC-1α-coregulated nuclear respiratory factor 1 (Nrf-1). Moreover, unlike 3-NP, the HD CAG repeat did not significantly alter mitochondrial pathways in STHdhQ111/Q111 cells, despite decreased Ppargc1a expression. Instead, the HD mutation enriched for processes linked to huntingtin normal function and Nf-κB signaling. Thus, rather than a direct impact on the mitochondrion, the polyglutamine tract may modulate some aspect of huntingtin's activity in extra-mitochondrial energy metabolism. Elucidation of this HD CAG-dependent pathway would spur efforts to achieve energy-based therapeutics in HD