Motoneuronal cell death is not correlated with aggregate formation of androgen receptors containing an elongated polyglutamine tract

Spinal and bulbar muscular atrophy (SBMA) is associated with an abnormal expansion of the (CAG)(n)repeat in the androgen receptor (AR) gene. Similar mutations have been reported in other proteins that cause neurodegenerative disorders. The CAG-coded elongated polyglutamine (polyGln) tracts induce the formation of neuronal intracellular aggregates. We have produced a model to study the effects of potentially 'neurotoxic' aggregates in SBMA using immortalized motoneuronal cells (NSC34) transfected with AR containing polyGln repeats of different sizes [(AR.Q(n = 0, 23 or 46)]. Using chimeras of AR.Q(n) and the green fluorescent protein (GFP), we have shown that aggregate formation occurs when the polyGln tract is elongated and AR is activated by androgens. In NSC34 cells co-expressing the AR with the polyGln of pathological length (AR.Q46) and the GFP we have noted the presence of several dystrophic neurites. Cell viability analyses have shown a reduced growth/survival rate in NSC34 expressing the AR.Q46, whereas testosterone treatment partially counteracted both cell death and the formation of dystrophic neurites. These observations indicate the lack of correlation between aggregate formation and cell survival, and suggest that neuronal degeneration in SBMA might be secondary to axonal/dendritic insults.     

Initial process of polyglutamine aggregate formation in vivo

BACKGROUND: Polyglutamine expansion in protein is responsible for several inherited neuro-degenerative diseases. The expansion has toxic effects on neural cells as well as results in forming aggregates. Using yeast, we examined the initial process of polyglutamine aggregate formation in vivo. RESULTS: Following expression, polyglutamine tracts were of a soluble form during a lag period, and then formed insoluble complexes. The lag was prolonged and the formation of insoluble complex became slower by decreasing the number of polyglutamine tracts and by a treatment with guanidine hydrochloride. Gel filtration analysis revealed that the soluble polyglutamine existed in a small form. Formation of polyglutamine aggregates appeared to follow similar kinetics reported in the in vitro studies, where polyglutamine tracts self-aggregate in a length-, concentration- and time-dependent manner. However, in vivo, Hsp104 was required for the conversion from a soluble to an insoluble state. Without Hsp104, polyglutamine tracts tended to remain in a small soluble form, prolonging the lag. Moreover, the dependency on Hsp104 for aggregate formation was strong with the short polyglutamine tract, and decreased with the long polyglutamine tract. CONCLUSION: For polyglutamine aggregate formation, a balance of parameters including the length of the polyglutamine tract, Hsp104, and level of polyglutamine expression determined its efficiency.     

Autophagy and polyglutamine diseases

In polyglutamine diseases, an abnormally elongated polyglutamine tract results in protein misfolding and accumulation of intracellular aggregates. The length of the polyglutamine expansion correlates with the tendency of the mutant protein to aggregate, as well as with neuronal toxicity and earlier disease onset. Although currently there is no effective cure to prevent or slow down the progression of these neurodegenerative disorders, increasing the clearance of mutant proteins has been proposed as a potential therapeutic approach. The ubiquitin-proteasome system and autophagy are the two main degradative pathways responsible for eliminating misfolded and unnecessary proteins in the cell. We will review some of the studies that have proposed autophagy as a strategy to reduce the accumulation of polyglutamine-expanded protein aggregates and protect against mutant protein neurotoxicity. We will also discuss some of the currently known mechanisms that induce autophagy, which may be beneficial for the treatment of these and other neurodegenerative disorders.     

The role of pre-existing aggregates in Hsp104-dependent polyglutamine aggregate formation and epigenetic change of yeast prions

Amyloid-like protein aggregates have been implicated in various diseases and in the protein-based inheritance of yeast prions. The molecular chaperone Hsp104 has been shown to be necessary for the aggregate formation of polyglutamine in yeast, and for the maintenance of several yeast prion phenotypes through the formation of self-propagating aggregates. In this paper, we show that the polyglutamine aggregates that are formed independently of Hsp104, are required for Hsp104 to efficiently produce more aggregates. Similarly, in the yeast prion [PSI+] system, Hsp104-dependent epigenetic changes to the [PSI+] prion phenotype require the presence of prion aggregates in the normal [psi-] state. We also show that the co-localization of different prion aggregates suggests that cross-seeding by different yeast prions increases the probability of Hsp104-dependent epigenetic change. These findings highlight the role of pre-existing aggregates in chaperone-dependent establishment of the epigenetic trait in yeast prions, and possibly in the pathology of several neurodegenerative diseases.     

Cell-autonomous and non-cell-autonomous toxicity in polyglutamine diseases

Polyglutamine diseases are neurodegenerative disorders caused by expansion of polyglutamine tracts in the coding regions of specific genes. One of the most important features of polyglutamine diseases is that, despite the widespread and in some cases ubiquitous expression of the polyglutamine proteins, specific populations of neurons degenerate in each disease. This finding has led to the idea that polyglutamine diseases are cell-autonomous diseases, in which selective neuronal dysfunction and death result from damage caused by the mutant protein within the targeted neuronal population itself. Development of animal models for conditional expression of polyglutamine proteins, along with new pharmacologic manipulation of polyglutamine protein expression and toxicity, has led to a remarkable change of the current view of polyglutamine diseases as cell-autonomous disorders. It is becoming evident that toxicity in the neighboring non-neuronal cells contributes to selective neuronal damage. This observation implies non-cell-autonomous mechanisms of neurodegeneration in polyglutamine diseases. Here, we describe cell-autonomous and non-cell-autonomous mechanisms of polyglutamine disease pathogenesis, including toxicity in neurons, skeletal muscle, glia, germinal cells, and other cell types.     

X-irradiation induces cell death in fetal fibroblasts

The impact of ionizing radiation on developing organisms has been widely studied for risk assessment purposes. Even though efforts have been made to decrease received doses to as low as reasonably achievable, the possibility of accidental exposure has to be considered as well. Mammalian gestation is usually divided into three periods. Radiation exposure during the 'pre-implantation period' may essentially result in embryonic mortality while exposure during the 'organogenesis period' may characteristically lead to malformations. In humans, the 'fetal period' is one of particular sensitivity to radiation induction of mental retardation, especially if the exposure occurs between weeks 8-15 of gestation. It is also admitted that prenatal irradiation may increase the risk of leukemia and childhood cancer, with an equal risk over the whole pregnancy. The aim of this study was to investigate the effects of moderate to high doses of X-irradiation on mouse skin fetal fibroblasts, one of the cell types subjected to the highest dose of radiation. Exposure of the cells to X-rays led to a rapid and significant increase in γ-H2AX foci, indicative of high levels of DNA double strand breaks. High doses (>2 Gy) also led to a pronounced G2-arrest and a decrease in the number of cells in the S phase, which was followed after 24 h by a decrease in cell survival and an increase in the level of apoptosis and necrosis. This study shows that mouse fetal skin fibroblasts are sensitive to high doses of X-irradiation. Furthermore, we report a better repair for higher doses than lower, which seems to indicate that little DNA damage is not necessarily repaired immediately. However, more sensitive approaches are necessary to identify the risk associated with low doses of radiation.     

Genetic modifiers of age-at-onset in polyglutamine diseases

Polyglutamine (polyQ) diseases are a group of clinically and genetically heterogeneous neurodegenerative diseases. Expansion size correlates with age-at-onset (AO) and severity, and shows a critical threshold for each polyQ disease. Although an expanded CAG tract is sufficient to trigger disease, not all variation in AO is explained by (CAG)_n length, which suggests the contribution of other modifying factors. Methods used to identify genetic modifiers in polyQ diseases have progressed from candidate genes to unbiased genome-wide searches. Inconsistency of results from candidate-genes studies are partly explained by sample size, study design and variable population frequency of “polymorphisms”; a genome-wide search may help elucidating more precise disease mechanisms underlying specific interaction networks. We review known genetic modifiers for polyQ diseases, and discuss developing strategies to find modulation, from common variants to networks disclosing small cumulative effects of key genes and modifying pathways. This may lead to a better understanding of genotype-phenotype correlation and the proposal of new potential targets for therapeutical interventions.     

Apoptosis-like cell death induction and aberrant fibroblast properties in human incisional hernia fascia

Incisional hernia often occurs following laparotomy and can be a source of serious problems. Although there is evidence that a biological cause may underlie its development, the mechanistic link between the local tissue microenvironment and tissue rupture is lacking. In this study, we used matched tissue-based and in vitro primary cell culture systems to examine the possible involvement of fascia fibroblasts in incisional hernia pathogenesis. Fascia biopsies were collected at surgery from incisional hernia patients and non-incisional hernia controls. Tissue samples were analyzed by histology and immunoblotting methods. Fascia primary fibroblast cultures were assessed at morphological, ultrastructural, and functional levels. We document tissue and fibroblast loss coupled to caspase-3 activation and induction of apoptosis-like cell-death mechanisms in incisional hernia fascia. Alterations in cytoskeleton organization and solubility were also observed. Incisional hernia fibroblasts showed a consistent phenotype throughout early passages in vitro, which was characterized by significantly enhanced cell proliferation and migration, reduced adhesion, and altered cytoskeleton properties, as compared to non-incisional hernia fibroblasts. Moreover, incisional hernia fibroblasts displayed morphological and ultrastructural alterations compatible with autophagic processes or lysosomal dysfunction, together with enhanced sensitivity to proapoptotic challenges. Overall, these data suggest an ongoing complex interplay of cell death induction, aberrant fibroblast function, and tissue loss in incisional hernia fascia, which may significantly contribute to altered matrix maintenance and tissue rupture in vivo.     

Involvement of apoptotic cell death in autoimmune diseases

A low rate of as well as a high rate of apoptotic cell death is involved in the development of various human autoimmune diseases. In rheumatoid arthritis (RA), impaired apoptosis of rheumatoid synovial cells appears to induce hyperplasia of the synovial tissues, whereas the acceleration of apoptotic cell death of osteoblasts may contribute to periarticular bone loss in patients with RA. Humoral factors including cytokines and growth factors present in the rheumatoid synovial tissues modulate the expression of apoptosis-related molecules in the cells, which inhibits or stimulates the apoptotic process of synovial cells and osteoblasts. In addition, investigations of animal arthritis models suggest that an enforced induction of apoptotic cell death of synovial cells ameliorates synovial tissue hyperplasia. The increase of salivary gland cell apoptosis and the resistance of apoptotic cell death in salivary infiltrating mononuclear cells have been observed in patients with Sj?gren's syndrome (SS). Immunohistochemical studies indicate that X chromosome linked inhibitor of apoptosis protein in salivary gland cells as well as Bcl-2/Bcl-xL in salivary infiltrating mononuclear cells may be critical anti-apoptogenic molecules in each cell type. Human T-lymphotropic virus type I (HTL V-I) is one of the pathogenic organisms for RA and SS, and we demonstrated that HTL V-I tax stimulates NF-kappa B nuclear translocation, inhibiting apoptotic cell death of human host cells, which may accelerate the autoimmune process. The association between the apoptosis of thyrocytes and the process of autoimmune thyroid diseases has also been examined, and our data suggest that Fas-mediated apoptosis of human thyrocytes is modulated by thyroid-stimulating antibodies, thyroid stimulation blocking antibodies, and cytokines. These data indicate that the correction of apoptotic cell death in each cell type will become a new therapeutic strategy for treatment of human autoimmune diseases.     

Involvement of polyglutamine endolysis followed by pyroglutamate formation in the pathogenesis of triplet repeat/polyglutamine-expansion diseases

The mechanism by which polyglutamine expansions in several proteins lead to neurodegenerative disorders remains largely unknown. The biochemical properties of polyglutamine repeats suggest one possible explanation; endolytic cleavage at a glutaminyl-glutaminyl bond followed by pyroglutamate formation may contribute to the pathogenesis through augmenting the catabolic stability, hydrophobicity, amyloidogenicity, and neurotoxicity of the polyglutaminyl proteins. The hypothesis points out novel therapeutic strategies to delay disease onset in genetically diagnosed presymptomatic patients.     

微RNA在多聚谷氨酰胺病中的作用研究进展

多聚谷氨酰胺(polyglutamine,PolyQ)病是一类因致病基因编码区的三核苷酸重复异常扩增而形成PolyQ肽链,最终导致选择性神经元变性乃至死亡的神经退行性疾病,其发病机制仍未完全阐明.微RNA(microRNA,miRNA)作为一种新的非蛋白质的基因表达调控因子,对真核生物的基因表达有非常重要的调控作用.近年来国内外学者日益关注miRNA在神经退行性疾病尤其是Po1yQ病中的功能及其作用机制.就目前miRNA在PolyQ病发病机制中的作用研究进展予以综述.     

An aggregation sensing reporter identifies leflunomide and teriflunomide as polyglutamine aggregate inhibitors

Intracellular protein aggregation is a common pathologic feature in neurodegenerative diseases such as Huntington' disease, amyotrophic lateral sclerosis and Parkinson' disease. Although progress towards understanding protein aggregation in vitro has been made, little of this knowledge has translated to patient therapy. Moreover, mechanisms controlling aggregate formation and catabolism in cellulo remain poorly understood. One limitation is the lack of tools to quantitatively monitor protein aggregation and disaggregation. Here, we developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). httQ72-Luc fails to aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein such as Q80-cfp. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity. Using httQ72-Luc with Q80(CFP/YFP) as seeds, we screened the Johns Hopkins Clinical Compound Library and identified leflunomide, a dihydroorotate dehydrogenase inhibitor with immunosuppressive and anti-psoriatic activities, as a novel drug that prevents polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis, since neither uridine treatment nor other pyrimidine biosynthesis inhibitors affected polyQ aggregation. Inducible cell line and cycloheximide-chase experiments indicate that these drugs prevent incorporation of expanded polyQ into an aggregate. This study demonstrates the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. As current trials are under-way for teriflunomide in the treatment of multiple sclerosis, we propose that this drug be considered a possible therapeutic agent for polyQ diseases.     

Inactivation of Drosophila Apaf-1 related killer suppresses formation of polyglutamine aggregates and blocks polyglutamine pathogenesis

Huntington's disease (HD) is caused by expansion of a polyglutamine tract near the N-terminal of huntingtin. Mutant huntingtin forms aggregates in striatum and cortex, where extensive cell death occurs. We used a Drosophila polyglutamine peptide model to assess the role of specific cell death regulators in polyglutamine-induced cell death. Here, we report that polyglutamine-induced cell death was dramatically suppressed in flies lacking Dark, the fly homolog of human Apaf-1, a key regulator of apoptosis. Dark appeared to play a role in the accumulation of polyglutamine-containing aggregates. Suppression of cell death, caspase activation and aggregate formation were also observed when mutant huntingtin exon 1 was expressed in homozygous dark mutant animals. Expanded polyglutamine induced a marked increase in expression of Dark, and Dark was observed to colocalize with ubiquitinated protein aggregates. Apaf-1 also was found to colocalize with huntingtin-containing aggregates in a murine model and HD brain, suggesting a common role for Dark/Apaf-1 in polyglutamine pathogenesis in invertebrates, mice and man. These findings suggest that limiting Apaf-1 activity may alleviate both pathological protein aggregation and neuronal cell death in HD.     

Adenovirus-mediated expression of mutant DRPLA proteins with expanded polyglutamine stretches in neuronally differentiated PC12 cells. Preferential intranuclear aggregate formation and apoptosis

To investigate the molecular mechanisms of neurodegeneration caused by expanded CAG repeats in dentatorubral-pallidoluysian atrophy (DRPLA), an autosomal dominant neuro degrees enerative disorder caused by unstable expansion of a CAG trinucleotide repeat in the DRPLA gene on 12p13.31, we established an efficient expression system for truncated and full-length DRPLA proteins with normal or expanded polyglutamine stretches in neuronally differentiated PC12 cells and fibroblasts using an adenovirus expression system. Although aggregate body formation was observed both in neuronally differentiated PC12 cells and in fibroblasts expressing truncated DRPLA proteins with Q82, >97% ( n = 3) of neuronally differentiated PC12 cells showed intra-nuclear inclusions, while only 31 21% ( n = 3) of fibro-blasts had intranuclear inclusions at 3 days after infection. The percentage of apoptotic cells was significantly higher in neuronally differentiated PC12 cells expressing the truncated DRPLA protein with Q82 than in fibroblasts, suggesting the possibility that intranuclear aggregate bodies are formed preferentially in neuronally differentiated PC12 cells and that these cells are more vulnerable than fibroblasts to the toxic effects of expanded polyglutamine stretches in the DRPLA protein. When the full- length DRPLA protein with Q82 was expressed, aggregate bodies were found exclusively in the nuclei of the neuronally differentiated PC12 cells, while they were found in the cytoplasm of fibroblasts. Despite the presence of aggregate bodies, apoptosis was not induced by expression of the full-length DRPLA protein with Q82 in either neuronally differentiated PC12 cells or fibroblasts, suggesting that the presence of intranuclear aggregate bodies is in itself not necessarily toxic to cells.      

Accumulation and aberrant modifications of alpha-crystallins in anterior polar cataracts

Crystallins are the major proteins found in the lens, and the localization of specific crystallins is well known. Overexpression and accumulation of alphaB-crystallin has been observed in response to stress conditions or in certain diseases, such as brain tumors and neurodegenerative diseases. The purpose of this study was to examine whether alpha-crystallins are modified during pathological myofibroblastic changes in lens epithelial cells. Lens epithelial cells attached to the anterior capsules of patients with nuclear or anterior polar cataracts were analyzed quantitatively for alpha-crystallin proteins and mRNAs using Western blot and RT-PCR analysis., respectively. The degree of modification of alpha-crystallins was determined by 2-dimensional gel electrophoresis followed by Western blotting. Higher molecular weight protein bands that were immunoreactive to anti-alphaA- and anti-alphaB-crystallin antibodies around 45 kDa accumulated more in the anterior polar cataract samples than in those with the nuclear type of cataracts. Also monomeric alphaB-crystallins accumulated more in lens epithelial cells of patients with anterior polar cataracts. By comparison, no significant changes were found in the levels of the mRNAs encoding alphaA- and alphaB-crystallins in the different types of cataracts. Both alphaA- and alphaB-crystallin proteins seemed to undergo more extensive modification in anterior polar cataracts. Conclusion. In addition to fibrotic changes, which accompany increased levels of extracellular matrix molecules, accumulation and abnormal modification of alpha-crystallins might be implicated in the pathogenic mechanism of this type of cataract.     

EdU induces DNA damage response and cell death in mESC in culture

Recently, a novel DNA replication precursor analogue called 5-ethynyl-2′-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.