Osmolytes modulate polyglutamine aggregation in a sequence dependent manner

Osmolytes stabilize protein structure and suppress protein aggregation. The mechanism of how osmolytes impact polyglutamine (polyQ) aggregation implicated in Huntington's disease was studied. By using a reverse‐phase chromatography assay, we show that methylamines‐trimethylamine N‐oxide and betaine are generic in enhancing polyQ aggregation, while a disaccharide trehalose and an amino acid citrulline moderately retard polyQ aggregation in a sequence specific manner. Despite the altered kinetics, the fundamental nucleation mechanism of polyQ aggregation and the nature of end stage aggregates remains unaffected. These results highlight the importance of using osmolytes as modulatory agents of polyQ aggregation.     

The composition of the polyglutamine‐containing proteins influences their co‐aggregation properties

The sequestration of crucial cellular proteins into insoluble aggregates formed by the polypeptides containing expanded polyglutamine tracts has been proposed to be the key mechanism responsible for the abnormal cell functioning in the so‐called polyglutamine diseases. To evaluate to what extent the ability of polyglutamine sequences to recruit other proteins into the intracellular aggregates depends on the composition of the aggregating peptide, we analysed the co‐aggregation properties of the N‐terminal fragment of huntingtin fused with unrelated non‐aggregating and/or self‐aggregating peptides. We show that the ability of the mutated N‐terminal huntingtin fragment to sequester non‐related proteins can be significantly increased by fusion with the non‐aggregating reporter protein [GFP (green fluorescence protein)]. By contrast, fusion with the self‐aggregating C‐terminal fragment of the CFTR (cystic fibrosis transmembrane conductance regulator) dramatically reduces the sequestration of related non‐fused huntingtin fragments. We also demonstrate that the co‐aggregation of different non‐fused N‐terminal huntingtin fragments depends on their length, with long fragments of the wild‐type huntingtin not only excluded from the nuclear inclusions, but also very inefficiently sequestered into the cytoplasmic aggregates formed by the short fragments of mutant protein. Additionally, our results suggest that atypical intracellular aggregation patterns, which include unusual distribution and/or morphology of protein aggregates, are associated with altered ability of accumulating proteins to co‐aggregate with other peptides.     

Solubilization and disaggregation of polyglutamine peptides

A method is described for dissolving and disaggregating chemically synthesized polyglutamine peptides. Polyglutamine peptides longer than about Q20 have been reported to be insoluble in water, but dissolution in--and evaporation from--a mixture of trifluoroacetic acid and hexafluoroisopropanol converts polyglutamine peptides up to at least Q44 to a form readily soluble in aqueous buffers. This procedure also has a dramatic effect on peptides which appear to be completely soluble in water, by removing traces of aggregate that seed aggregation. The protocol makes possible solution studies-including in vitro aggregation experiments--on polyglutamine peptides with repeat lengths associated with increased risk of Huntington's Disease and other expanded CAG repeat diseases. It may also be useful in conducting reproducible, quantitative aggregation studies on other polypeptides.     

Inducing huntingtin inclusion formation in primary neuronal cell culture and in vivo by high-capacity adenoviral vectors expressing truncated and full-length huntingtin with polyglutamine expansion

BACKGROUND: Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Vector-mediated delivery of N-terminal fragments of mutant htt has been used to study htt function in vitro and to establish HD models in rats. Due to the large size of the htt cDNA vector-mediated delivery of full-length htt has not been achieved so far. METHODS: High-capacity adenoviral (HC-Ad) vectors were generated expressing mutant and wild-type versions of N-terminal truncated and full-length htt either in vitro in primary neuronal cells or in the striatum of mice. RESULTS: In vitro these vectors were used for transduction of primary neuronal cells isolated from E17 mouse embryos. Expression of mutant htt resulted in the formation of htt inclusions, a surrogate marker of the HD pathology. Kinetics of generation and localization of htt inclusions differed between truncated and full-length htt carrying identical mutations. Following injection into the striatum vector-mediated expression of mutant truncated htt led to prominent accumulation of htt inclusions in cell nuclei, while inclusions formed upon expression of mutant full-length htt localized to the cytoplasm. CONCLUSIONS: These results indicate that HC-Ad vector-mediated in vitro and in vivo delivery of truncated and full-length mutant htt results in prominent inclusion formation in neuronal cells but in different cell compartments. These vectors will be useful tools for studying HD and may be used to generate large animal HD models.     

Towards humane end points: behavioural changes precede clinical signs of disease in a Huntington's disease model

The number of animals used in science is increasing, bringing a concomitant obligation to minimize suffering. For animals with progressive conditions, euthanasia at a 'humane end point' is advised if the end point is scientifically valid, predictive and accurate. Our aim was to test the hypothesis that behavioural changes would reliably precede clinical signs of disease in a progressive neurological model, using retrospective analysis. We observed 100 pair-housed female R6/1 transgenic Huntington's disease (HD) mice and 28 pair-housed female wild-type (WT) mice in standard- or resource-enriched cages. Disease progression was monitored until one member of each HD pair reached a pre-defined end point based on pathological symptoms (HD end). This mouse was then euthanized together with its cage mate (HD other) and any matched WT pairs. At euthanasia, HD mice had significantly greater absolute and relative organ weights, and significantly higher alpha1 acid glycoprotein concentrations than WT mice, indicating reduced welfare. HD mice initially showed significantly greater use of cage resources than WT mice but this declined progressively. Steeper declines, and earlier cessation, in the use of some climbing and exploration resources occurred in the HD end mice compared with the HD other mice. Behavioural change can be an early indicator of disease onset.     

Neurodegenerative processes in Huntington's disease

Huntington''s disease (HD) is a complex and severe disorder characterized by the gradual and the progressive loss of neurons, predominantly in the striatum, which leads to the typical motor and cognitive impairments associated with this pathology. HD is caused by a highly polymorphic CAG trinucleotide repeat expansion in the exon-1 of the gene encoding for huntingtin protein. Since the first discovery of the huntingtin gene, investigations with a consistent number of in-vitro and in-vivo models have provided insights into the toxic events related to the expression of the mutant protein. In this review, we will summarize the progress made in characterizing the signaling pathways that contribute to neuronal degeneration in HD. We will highlight the age-dependent loss of proteostasis that is primarily responsible for the formation of aggregates observed in HD patients. The most promising molecular targets for the development of pharmacological interventions will also be discussed.     

Study on the variation in X-ray sensitivity of human diploid skin fibroblasts. Normal radiosensitivity of cells derived from patients with Huntington's disease

9 cell strains derived from patients with Huntington's disease and 9 from age- and sex-matched controls were investigated for X-ray sensitivity. No differences in radiosensitivity were observed for the two groups. The two groups taken together reveal a dependence of radiosensitivity on intrinsic cloning efficiency which in turn correlates with donor age. A difference in radiosensitivity between males and females is also indicated although at the borderline of significance. As a parameter for radiosensitivity the dose needed to obtain 0.1% survival appears superior to the Do.     

Iron dysregulation in Huntington's disease

Huntington's disease (HD) is one of many neurodegenerative diseases with reported alterations in brain iron homeostasis that may contribute to neuropathogenesis. Iron accumulation in the specific brain areas of neurodegeneration in HD has been proposed based on observations in post‐mortem tissue and magnetic resonance imaging studies. Altered magnetic resonance imaging signal within specific brain regions undergoing neurodegeneration has been consistently reported and interpreted as altered levels of brain iron. Biochemical studies using various techniques to measure iron species in human samples, mouse tissue, or in vitro has generated equivocal data to support such an association. Whether elevated brain iron occurs in HD, plays a significant contributing role in HD pathogenesis, or is a secondary effect remains currently unclear.

     

Creatine therapy provides neuroprotection after onset of clinical symptoms in Huntington's disease transgenic mice

While there have been enormous strides in the understanding of Huntington's disease (HD) pathogenesis, treatment to slow or prevent disease progression remains elusive. We previously reported that dietary creatine supplementation significantly improves the clinical and neuropathological phenotype in transgenic HD mice lines starting at weaning, before clinical symptoms appear. We now report that creatine administration started after onset of clinical symptoms significantly extends survival in the R6/2 transgenic mouse model of HD. Creatine treatment started at 6, 8, and 10 weeks of age, analogous to early, middle, and late stages of human HD, significantly extended survival at both the 6- and 8-week starting points. Significantly improved motor performance was present in both the 6- and 8-week treatment paradigms, while reduced body weight loss was only observed in creatine-supplemented R6/2 mice started at 6 weeks. Neuropathological sequelae of gross brain and neuronal atrophy and huntingtin aggregates were delayed in creatine-treated R6/2 mice started at 6 weeks. We show significantly reduced brain levels of both creatine and ATP in R6/2 mice, consistent with a bioenergetic defect. Oral creatine supplementation significantly increased brain concentrations of creatine and ATP to wild-type control levels, exerting a neuroprotective effect. These findings have important therapeutic implications, suggesting that creatine therapy initiated after diagnosis may provide significant clinical benefits to HD patients.     

Unaided trifluoroacetic acid pretreatment solubilizes polyglutamine peptides and retains their biophysical properties of aggregation

Understanding the biophysical mechanism of polyglutamine (polyGln) aggregation is important to unravel the role of aggregates in the pathology of polyGln repeat disorders. To achieve this, synthetic polyGln peptides are widely used. Their disaggregation and solubilization is essential because it plays a crucial role in reproducing biophysical experimental data under in vitro conditions. Pretreatment with trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP) at a 1:1 ratio is currently the method of choice to achieve solubility of polyGln peptides. Here we report that the disaggregation and solubilization of polyGln peptides can be achieved by TFA alone. We tested TFA due to the close similarity of it with HFIP in the nature of H-bond breakage and formation, higher cost, and the problems faced by us in the availability of HFIP. Our results demonstrate that the TFA disaggregated polyGln sequences give similar solubilization yield, aggregation kinetics, thioflavin T (ThT) binding, and structural features in comparison with the TFA/HFIP method. Furthermore, we show by limited validation studies that the proposed TFA method can replace the existing TFA/HFIP disaggregation method of polyGln sequences.     

Responses of Huntington's disease and ataxia telangiectasia lymphoblastoid cells to bleomycin

Ionizing radiation sensitive, mutant human lymphoblastoid cell lines derived from patients with Huntington's disease (HD), or ataxia telangiectasia (AT) both showed cross sensitivity to bleomycin, as assayed by reduced cell viability and increased frequency of chromosome aberrations compared to normal controls. In contrast to AT cells which failed to show inhibition of DNA synthesis after exposure to ionizing radiation, or bleomycin treatment, the sensitive cells from HD patients had depressed rates of DNA synthesis after damage with these agents, similar to that seen in normal cells. In terms of progression through the cell cycle bleomycin damaged AT cells moved from G1 into S and from S to G2 + M at almost the same rate as untreated cells. Bleomycin treated HD cells showed a large proportion of cells blocked in G1, cells were slowed down in S, the rate of entry to G2 + M was reduced and only 5% of cycling cells reached G2. Progress through the cell cycle in normal cells exposed to bleomycin showed a partial block in G1 and the rate of entry to G2 + M was reduced. These differences in response of normal, AT and HD cells to ionizing radiation and bleomycin treatment indicates that the defect underlying the sensitivity is different in HD cells from that in AT cells.     

Shaping the role of mitochondria in the pathogenesis of Huntington's disease

Intense research on the pathogenesis of Huntington's disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, revealed multiple potential mechanisms, among which mitochondrial alterations had emerged as key determinants of the natural history of the disease. Pharmacological and genetic animal models of mitochondrial dysfunction in the striatum, which is mostly affected in HD corroborated a key role for these organelles in the pathogenesis of the disease. Here, we will give an account of the recent evidence indicating that the mitochondria-shaping machinery is altered in HD models and patients. Since its correction can counteract HD mitochondrial dysfunction and cellular damage, drugs impacting on mitochondrial shape are emerging as a new possibility of treatment for this devastating condition.     

Defining the role of the Bcl-2 family proteins in Huntington's disease

B-cell lymphoma 2 (Bcl-2) family proteins regulate survival, mitochondria morphology dynamics and metabolism in many cell types including neurons. Huntington''s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat tract in the IT15 gene that encodes for the protein huntingtin (htt). In vitro and in vivo models of HD and HD patients'' tissues show abnormal mitochondrial function and increased cell death rates associated with alterations in Bcl-2 family protein expression and localization. This review aims to draw together the information related to Bcl-2 family protein alterations in HD to decipher their potential role in mutated htt-related cell death and mitochondrial dysfunction.     

Changes in cardiac nucleotide metabolism in Huntington's disease

ABSTRACT

Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.